RECOMBINANT DNA TECHNOLOGY

60.457

LAB MANUAL

2003
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TABLE OF CONTENT page

Schedule 3
General Instructions 4
WHMIS 10
Introduction 12

Procedures
Lab 1: Screening of a lambda genomic library for a particular gene 19
Part I: Titration of genomic library
Part II: Plating of genomic library
Part III: Chemiluminenscent DIG-labeled hybridization
Part IV: Selection of positive clone

Lab 2: Subcloning of genomic DNA fragment into pM13 plasmid 25

Part I: Preparative restriction enzyme digestion of genomic
library clone and purification of genomic fragment
Part II: Preparation of pM13 plasmid DNA
Part III: pM13 plasmid restriction enzyme digestion
Part IV: Ligation of plasmid and genomic fragment
Part V: Transformation of E. coli and selection of recombinants
Part VI: Verification of recombinant by sizing uncut plasmid DNA (QIAprep)
on agarose gel

Lab 3: Restriction enzyme mapping of genomic fragment subcloned 35

in pM13
Part I: Preparation of recombinant pM13 subclone DNA
Part II: Restriction enzyme mapping (single digests)

Lab 4: DNA Sequencing of genomic fragment subcloned in pM13 38

Part I: Sequencing reaction of recombinant plasmid DNA
Part II: Gel electrophoresis of DNA sequencing reactions and autoradiograph

Appendix
Titer Sample Calculation 44
Media and Solutions 45
Equipment Operation
Pipetman operation 47
Agarose gel electrophoresis 49
Floor model centrifuge operation 50
References 52

Sample Lab Exam 53

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RECOMBINANT DNA TECHNOLOGY 60.457 SCHEDULE - 2003

Date Week # Lab # Experiments

______________________________________________________________________________
Jan 13 1 1 Part I: Titration of genomic library

Jan 20 2 1 Part II: Genomic library blot preparation

Jan 27 3 1 Part III: Plaque Hybridization

2 Part I: (A) Preparative restriction enzyme digestion
of genomic library clone

Feb 3 4 1 Part IV: Selection of Positive Clone

2 Part I: (B) Purification of genomic fragment
2 Part II: DNA preparation of pM13 plasmid
3 Part I: DNA preparation of recombinant pM13 subclone

Feb 10 5 2 Part III: (A) pM13 plasmid restriction enzyme digestion

(B) Agarose gel electrophoresis of plasmid DNA
and genomic insert

Feb 17 6 MID-TERM BREAK

Feb 24 7 2 9:00 am Part IV: Ligation of plasmid and genomic fragment

2 Part V: Transformation of E. coli and selection of
recombinants

Feb 28 7 2 Part VI: Verification of recombinant by sizing of

rapid DNA plasmid preps on agarose gel
-streak colonies on LB-AMP

Mar 3 8 2 Part VI: Verification of recombinant by sizing of

rapid DNA plasmid preps on agarose gel
-isolate plasmid DNA/gel electrophoresis
3 Part II: Restriction enzyme mapping (single digests)

Mar 10 9 4 Part I: Sequencing reaction of recombinant plasmid DNA

4 Part II: Gel electrophoresis of DNA sequencing reactions and
autoradiograph

Mar 17 10 4 Part III: Analysis of sequencing gel results & Lab Exam Oultine

Mar 24 11 Lab exam Prep Question Period

Mar 31 LAB EXAM

LAB REPORT DUE DATES - Plan ahead.

Keep up with lab report write up especially for labs 2 and 3 as they are handed in on the same
day. Both reports take considerable time, especially report 2 as data was accumulated over a
month and half time period. Data is available on your website shortly after collected.
Feb 24 Lab 1 report due
Mar 17 Lab 2 report due
Mar 17 Lab 3 report due
Mar 24 Lab 4 report due
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GENERAL INSTRUCTIONS
Instructor: Dr. L. Cameron Office: 414B
Demonstrators: Prashen Chelikani Lab: 410 Buller

Lab Location: 201 Buller

Website:
www.umanitoba.ca/faculties/science/microbiology/staff/cameron/
Information available at the website: lab manual, changes/corrections, additional information,
data, marks
REGULATIONS
1. Students must wear a lab coat. There is no smoking, drinking, or eating in the lab.
2. Students work in pairs.

EVALUATION 60.457
1. The lab is worth 20% of the final mark: 8% for lab reports and 12% for lab exam.
2. You must pass the lab to pass the course (10/20%).
3. The lab exam takes place during scheduled lab period (refer to schedule for date). The
lab exam is 1.5 hours and must be written in pen. An example lab exam is available in
the lab manual appendix.
4. Lab reports are to be handed in as stated in schedule by 4:30 pm of that day. Hand in
lab reports through slotted drawer in room 414 ONLY. Instructor and demonstrators do
not accept lab reports. If handing in lab late, 1 mark will be subtracted for each class day
late. Marked lab reports will be returned to students the next week. A late report will not
be accepted after that report has been returned to the class.
5. Lab report marks are final unless an obvious error in addition of marks has been made.
However, if a student feels they have a legitimate complaint, please direct attention to lab
instructor.
6. Approximately two weeks prior to the lab exam, a brief outline of lab exam format and
information content will be presented available on the website.
7. You must notify the lab instructor no later than two school days after the missed lab
exam of your intent to write a deferred lab exam. The deferred lab exam must be
rescheduled before the end of this term’s classes. Failure to comply will result in a zero
on your lab exam.
8. Plagiarism (copying another student’s lab report (present or previous year) or
copying published literature without citing is a violation of University regulations.
Refer to the STUDENT DISCIPLINE BY-LAW in your student handbook (rule
book) for action taken for plagiarism.

LAB REPORT PRESENTATION

1. A reference file is available in the science library (1 hour reserve).
2. Lab reports must be written in pen (no pencil) or typed (preferred).
3. Number pages.
4. On the front page of the report state:
• Course name and number
• Experiment number and Title
• Group # and section #
• Individual or Group name(s). If handing in an individual report, also include lab
partners name.
• GROUP report or INDIVIDUAL report
• Date
3. Lab report information is to be presented exactly as requested in lab manual. Number
 5

sections the same as the lab manual.

4. Lab report may be done as an individual effort or a group effort by the two students that
carried out the experiment. One report or two reports may be handed in per group. The
decision on the number of reports per group is totally dependent on members of the
group. This decision may be changed any time during the term. Therefore for each lab
report the group has the option to hand in one or two reports exclusive of what has been
done before or after that particular report. Indicate on the front page of the report if
the report is a group report or an individual report. If handing in an individual report
also include lab partner’s name.

5. Always include a sample of each type of calculation in your lab report.

6. If a group’s data is not workable, borrow data from another group and reference. Non
workable refers to data that cannot be plotted, used for calculations or required analysis.
It does not necessarily mean the expected data.

7. Cite reference in text of lab report and record full reference at end of lab report. When
should you cite and reference. The following is a good definition of plagiarism that
explains when you should cite a reference. “The unacknowledged use of another
person’s work, in the form of original ideas, strategies, and research, as well as
another person’s writing, in the form of sentences, phases and innovative
terminology.” (Spatt1, 1983, p.438) This is done by using bracketed reference number
that you used when listing references at end of lab report or by bracketing first authors
name and date. Quote text unless you paraphrase completely in your own words. But
remember, quotes should only be a small part of your work. If you are using the name
year system, list the references alphabetically. Some examples are as follows (McMillan2
1997):

Binder V. Hendriksen C, Kreiner S. 1985. Prognosis in Crohn’s disease - - based on

results from regional patient group from county of Copenhagen. Gut 26:146-50.
Danforth DN, editor. 1982. Obstetrics and gynecology. 4th ed. Philadelphia: Harper and
Row. 1316 p.
Petter JJ. 1965. The lemurs of Madagascar. In: DeVore I, editor. Primate behavior:
field studies of monkeys and apes. New York: Holt, Rinehart and Winston. p 2920319.

If available only on the web:

Kingsolver JC, Srygley RB. Experimental analyses of body size, flight and survival in
pierid butterflies. Evol. Ecol. Res. [serial online] 2000;2:593-612. Available from:
Colgate University online catalog. Accessed 2000 Oct 3.

8. Personal or Professional Electronic sources2:

Cite in-text by putting the following in parentheses, author’s last name or file name (if
no author’s name is available) and publication date or the date of access (if no
publication date is available).
At the end of report list
(i) author or organization
(ii) publication date or date last revised

1
Spatt, B. (1983). Writing from Sources. New York: St. Martin’s Press.
2
McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston:
Bedford Books: 1997. 197 p. and McMillan, V.E. 2001. Writing Papers in the Biological
Sciences. 3rd ed. Boston: Bedford Books. 123 p.
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(iii) title of Web site

(iv) URL site in angle brackets
(v) the date accessed.

Cameron, L. 60.344 Microbial Physiology Lab Information

<http://www.umanitoba.ca/faculties/science/microbiology/staff/cameron/60_344.htm>.
Accessed 2002 April 12.

Table presentation
• Table number and title (legend) presented above the table body.
• Number tables using arabic numbers, even if only one table in a report.
• Include enough information in title to completely describe table, eliminating the necessity
to search elsewhere in the lab report to understand information presented in table. Table
title starts with an incomplete sentence. Additional complete sentences may be included
to adequately describe the table (this also applies to figures).
• If abbreviations are used in table, indicate what abbreviations mean as a footnote. Other
footnotes may be required to clarify material in the table.
• Like information should be in columns making it easier to view the table.
• Data in columns should be listed under the centre of each heading. Align decimal points
and dashes. If a number value is less than 1 always include zero before the decimal.
• Column or Row headings should be complete and self explanatory. A heading is a
separate entity from the title. It cannot be assumed information given in the title is
adequate for a heading. The unit of measurement should only be included in the heading,
not in column data.
• Group related column headings under larger headings.
• If information is the same for each column or row do not include but treat as a footnote.
• Make the table as concise as possible but include all necessary information. For
example, any constant experimental conditions that would change the data presented.
• Tables should be properly set up with a straight edge. Horizontal lines must be included
but it not necessary to always include vertical lines.
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Figure presentation (graphs, diagrams, photographs, films)

• Figures are to be numbered separate from tables, using arabic numbers. Include figure
number even if only one figure.
• Figure number and figure legend should be presented below the graph. The figure
legend, like the table, starts with an incomplete sentence describing the graph. Do not
repeat just the labels of the x- and y-axis but present in a descriptive manner. Additional
sentences should be included if additional information is required to completely describe
figure, for example, any constant experimental conditions that affect the data presented.
• All diagrams, photographs, and films are figures and should be completely labelled.
• For figures of graphs, there is one dependent variable plotted and one or more
independent variables plotted. The dependent variable is a function of the independent
variable. It is accepted practise to plot the independent variable on the x-axis and the
dependent variable on the y-axis. For example the measurement of absorbance
(dependent) with increasing concentration of protein (independent). The size of the
graph should fit the plot(s). The axis should not necessarily start at zero. Place graph
completely within graph grid, this includes axis labels and legend. The overall size of
graph should not be too large but should not be so small that information is obscured.
The graph must be completely labelled (always include units). Use different symbols for
each plot (not different coloured pens) on a graph. If more than one plot, explain
symbols in legend or in a key included in the body of the graph. Graph plots can be
drawn in a number of ways (this depends on the plot): (a) best fit straight line, (b) join
each point with a straight line, and (c) use a flexible curve ruler or french curve. Do not
drawn a free hand line.

Note: When writing your lab reports you are frequently requested to present both a table and a
figure for a given set of data, similar to keeping a research journal. This is not the accepted
practice for papers published in journals or books. Usually either a table or a figure is presented
for a given set of data and depending on nature of data, it may only be summarized in the text.
How do you make a choice of data presentation? The aim is to effectively and efficiently
demonstrate what you want to show, for example, correlations, comparisons, pattern, trends, etc
(McMillan 1997).

REFERENCE

McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston: Bedford
Books: 1997. 197 p.
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LAB STANDARD OPERATIONS PROCEDURE (SOP)

Bench area: Wash bench area before and after use with AIRx109.

Personal safety: You must wear a lab coat. Wear coat only in the lab, transport separately outside
of the lab (in a plastic bag). Wash hands with antibacterial soap before leaving the lab. No eating or
drinking in the lab. Use aseptic technique for transfer of bacteria. This is to protect yourself as
much as to ensure the purity of your culture. Protect hands with gloves and eyes with glasses when
needed. The gloves provided in the lab are to be disposed of after use.

Biohazards: Know biosafety risk groups. Handle all cultures as potential pathogens. Never mouth
pipette. Always use a pro-pipette. If you spill a culture, cover the spill with paper towels. Pour
AIRx109 over the towels to saturate. Gather up soaked towels and discard. Wipe area to dryness
with fresh paper towels. Wash hands with soap and water. Place cultures on discard trolley. All
cultures are autoclaved before disposing. Dispose of eppendorf tubesa in petri plate containers.
Dispose of pipetman tipsa in clear plastic lined basins along with glass or plastic Pasteur pipets,
broken glassware, glass slides, brittle plastic objects, metal objectsa (not needles or blades). Bacteria
dilutions may to be poured down the sink and the tubes rinsed before placing on the discard trolley.
Rinse sink with lots of water.
When handling level 2 microorganisms you must wear disposable gloves, make sure any cuts on
your hands are covered with a bandage, and be aware of the possibility of bacteria aerosol when you
flame your loop.
a
due to the multi-use nature of the teaching lab, all eppendorf tubes, pipetman tips, Pasteur pipets,
brittle plastic or metal objects will be treated the same as similar items contaminated with
microorganisms.

Glassware (unbroken): Remove tape and pen markings (use alcohol) from glassware before
placing on discard trolley. Used glassware should be rinsed and placed on the discard trolley.
Rinsed test tubes should be placed in tray provided on the discard trolley. Used glass pipettes
should be placed in pipette holders.

Petri plate culture and non-sharps solid culture material disposal: use covered plastic containers
lined with clear plastic bags for contaminated petri dishes or any bacteria contaminated solid non-
sharps material (eppendorf tubes, API strips, antibiotic strips, microtitration plates, etc)

Hazardous material disposal: Examples: radioactive material, ethidium bromide, sovents, etc. The
lab demonstrator will instruct proper disposal methods for labs that contain hazardous materials.
These materials must be disposed of in appropriately labelled containers and disposed via the safety
office. Use fumehood when recommended. A MSDS binder available in lab gives information on
all hazardous materials used in the lab. Use extreme care with flammable solvents. Alcohol used to
flame spread rod should never be positioned within 40 cm of flame. Never put a very hot spread rod
into a beaker of alcohol. The alcohol may catch fire. Many of the immunochemicals are preserved
in 0.1% Na azide...handle with gloved hands. Handle caustic (acids and bases) solutions with care.
Never discard an acid or base greater than one molar down the sink. Discard in labelled glass
containers provided. Use lots of water when discard caustic solutions (< 1M). These materials are
disposed of through the university safety office. Never pour solvents down the sink (eg. phenol,
ether, chloroform, etc). Discard in labelled containers provided.
Ethidium Bromide: This chemical is a potent carcinogen and must be handled with extreme care.
Always handle with disposable gloves.
Phenol: Phenols rapidly attack the skin and cause serious burns although poisoning by absorption
through the skin may occur even though the skin surface has remained intact. Wash well with water
if skin is splashed. If phenol spills occur, mop up with ethanol.

Sharps disposal: Dispose of all sharps (needles, syringes, razors, scalpel blades) in specified
container. Dispose of syringe with needle attached - do not take apart. Do not replace the needle
cap before disposing (high frequency of accidents occur when replacing cap). Sharp’s containers are
autoclaved before disposing. .
 9

Broken glass disposal: Dispose of broken glass in labelled plastic containers lined with clear
plastic. Transferred to boxes before discarding.

Know location: Exits, fire extinguisher, eye wash, sink shower, and first aid kit. This
information is given in the first pre-lab.

Equipment operation: Know how to operate equipment before use. DO NOT use equipment
unless you know exactly how to operate the equipment. The demonstrator is always available to
assist. Please follow instructions in appendix for proper clean up of Spectronic 20D. Ensure the
spec tubes are thoroughly washed and rinsed with distilled water before replacing in rack upside
down as you (hopefully) found the tubes.

Leave your bench area clean All equipment and supplies should be returned to original
location.

LABORATORY BIOSAFETY GUIDE

Although, only Level 1 bacteria risk group is used in this lab, level 2 bacteria are used by other
labs in this room. Follow standard operation procedures, SOP (see above).

The University of Manitoba Biosafety Guide (Feb 2000) and Health Canada Laboratory
Biosafety Guidelines booklets are available in your lab. Biosafety information is also available
at the Health Canada websites:
Guidelines: http://www.hc-sc.gc.ca/hpb/lcdc/biosafty/docs/index.html
MSDS (infectious agents): http://www.hc-sc.gc.ca/hpb/lcdc/biosafty/msds/index.html
There is no listing of level 1 agents in the guidelines or MSDS pamphlets
Risk group 1 bacteria are low individual and community risk and are unlikely to cause disease
in healthy workers.
Risk group 2 bacteria are moderate individual risk and limited community risk. Bacteria in this
group can cause human or animal disease but are unlikely to infect healthy laboratory workers.
Effective treatment is available. Risk of spreading is limited.

CONTAINMENT LEVEL 1 (UM biosafety guide p. 11)

• microbiology lab with washable walls, countertops and hand wash sink
• established safe laboratory practices (hand washing and disinfection of countertops)
• general WHMIS safety training
• UM lab registration

CONTAINMENT LEVEL 2 (UM biosafety guide p.11)

• all of level 1 specifications
• biosafety permit
• biological safety cabinet (not required)
• biohazard sigage
• a written standard operations procedure
• MSDS for the infectious agent
 10

WHMIS

The Workplace Hazardous Materials Information System (WHMIS) is a system for safe
management of hazardous materials. WHMIS is legislated by both the federal and provincial
governments.

Under WHMIS legislation, laboratories are considered to be a workplace, and students are
workers. By law, all workers must be familiar with the basic elements of the WHMIS system.

The WHMIS program includes:

1. Cautionary labels on containers of controlled products. Consumer products, explosives,
cosmetics, drugs and foods, radioactive materials, and pest control products are regulated
separately, under different legislation.
2.Provision of a Material Safety Data Sheet (MSDS) for each controlled product
3.A worker education program

1. A. SUPPLIER LABELS

Controlled products must have a label of prescribed design which includes the following
information:
PRODUCT IDENTIFIER - trade name or chemical name
SUPPLIER IDENTIFIER - supplier's name and address
MSDS REFERENCE - usually, "See MSDS supplied"
HAZARD SYMBOL - (see illustration on next page)
RISK PHRASES - describes nature of hazards
PRECAUTIONARY MEASURES
FIRST AID MEASURES

B. WORKPLACE LABELS

All material dispensed in a workplace container must be labelled with the Product Name,
Precautionary Measures (simplified) and Reference to Availability of MSDS.

2. MSDS

Individual course MSDS are located in a binder in your lab (Room 201 binder located in 204).
The main MSDS binders are located in the Microbiology preparation room, 307/309 Buller.
MSDS are also available on the local area computer network (see your demonstrator, if
necessary).

The MSDS will provide: relevant technical information on the substance, chemical hazard data,
control measures, accident prevention information, handling, storage and disposal procedures,
and emergency procedures to follow in the event of an accident.

3. SAFETY

The Laboratory Supervisor will provide information on the location and use of safety equipment,
and emergency procedures.
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WHMIS CHART
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INTRODUCTION

OBJECT

The object of this lab is to learn recombinant DNA techniques. In this lab the highly
conserved calmodulin gene of the water mould, Achlya, is investigated. As the study of Achlya
calmodulin gene has already been documented at the molecular genetics level (LeJohn, 1989), it
presents a system that allows us to perform the isolation of the Achlya calmodulin library clone,
and the preparation and study of the plasmid subclone in a short period of time.

GENOMIC LIBRARY PREPARATION

The genomic library was prepared by partially digesting Achlya genomic DNA with the
restriction enzyme Mbo1. The 20 kb genomic DNA fragments were collected by sucrose
centrifugation and ligated into the BamH1 restriction site of EMBL3 lambda arms (Sambrook et
al, 1989). The EMBL3 arms containing Achlya genomic DNA were then in vitro packaged in
lambda. This is the Achlya lambda library. With cloning approximately 20 kb genomic
fragments into lambda arms, this library requires approximately10000 plaques to represent 1
complete library (genome). This amount of plaques can be plated on one 150 mm diameter petri
plate. The Achlya genomic library was screened using random primer labelled plasmid pCM116
which contains the electric eel calmodulin cDNA. Once a pure positive clone for the Achlya
calmodulin gene is obtained, large scale preps of the calmodulin clone lambda DNA is prepared.
The lambda DNA is digested with numerous restricition enzyme (must be located in the multi-
cloning site of pM13). The restriction digests are run on an agarose gel and a southern blot is
prepared. The southern blot is screened with random primer labelled plasmid pCM116 which
contains the electric eel calmodulin cDNA to find the smallest DNA restriction fragment that
contains the complete coding region of the calmodulin gene The ideal size is two to four kb.
This allows not only sequencing of the calmodulin gene but permits characterization of the
upstream and downstream gene signals.

PLASMID pM13
The plasmid pM13 (3 kb) (Figure 2) will be used throughout this lab. The diagram is
schematic, not drawn to scale. Plasmid pM13 is a derivative of pUC, which retains the
ampicillin resistant gene and the Col E1 ori (relaxed origin of replication). The origin of
replication allows autonomous replication in E. coli. The ampicillin resistant gene is a marker
gene for the presence of the plasmid (growth on media containing ampicillin).

The plasmid was constructed by inserting a portion of the Lac Z gene (remainder found on
the F’ factor - delta M15 Lac gene) and then inserting T3 and T7 promoters into the coding
region of the Lac Z gene. The two lac genes complement each other to give a functional $-
galactosidase gene. The Lac Z gene permits detection of recombinant by blue/white selection of
bacterial colonies on plates containing X-gal/IPTG. The Lac Z genes codes the enyzme $-
galactosidase. The non-inducing histochemical stain, Xgal (5-bromo-4-chloro-3-indolyl-$-D-
galactoside) is a substrate of $-galactosidase. IPTG (isopropropyl-$-D-thiogalactoside) is a non-
metabolizable inducer of $-galactosidase. Colonies containing a plasmid with an intact Lac Z
gene produce $-galactosidase. $-galactosidase cleaves Xgal resulting in the production of blue
colonies. Colonies containing a recombinant plasmid with an interrupted Lac Z gene do not
have a functional $-galactosidase gene therefore the colonies remain translucent or white (color
of medium). An additional factor to consider is the delta M15 lac gene carried on the F'
episome. Host bacteria that have lost the F' episome will produce translucent/white colonies on
the X-gal/IPTG agar plates. This occurs at a frequency of 1 in 100.
 13

Figure 1: Plasmid pM13 multi-cloning site

 14

Unique restriction sites were inserted (multiple cloning site) between the two promoters.
DNA sequencing primer sites overlap the multiple cloning site and T3 and T7 promoters. The
T7 promoter side has DNA sequencing primers; T7 primer, M13 20 primer and SK primer while
the T3 promoter side has the primers; T3 primer, KS primer and reverse primer. The choice of
sequencing primers available at both the T3 and T7 promoter side of the insert allows for
flexibility (3 choices of primers for sequencing the insert in both directions). The two
promoters, T3 and T7, allow RNA transcripts to be synthesized from inserts cloned in the multi
restriction sites. The RNA transcripts can be sequenced using reverse transcriptase and translated
using an in vitro translation system.

In summary, these features of pM13 permit gene cloning, restriction mapping, nested deletion
construction, site specific mutagenesis, generation of RNA transcripts, and DNA sequencing. In
this lab the pM13 plasmid will be used for cloning, restriction mapping and DNA sequencing.

RESTRICTION ENZYMES (TYPE II)

Restriction enzymes recognize a specific target sequence (tetra, penta, hexa, or

heptanucleotides which have an axis of rotational symmetry) in double stranded DNA and make
blunt ended or staggered cuts of the polynucleotide chains. The classic example of a restriction
enzyme is Eco RI that cleaves in a staggered manner generating short single-stranded sticky
ends. The cleavage sites are located on either side of a short palindromic sequence that is part of
the enzyme recognition site.

5'.................G A A T T C ......................3'

3'.................C T T A A G.......................5'

EcoR1 cleaves recognition site

indicated by arrows to produce
following fragments

5'..............G + A A T T C..............3'
3'..............C T T A A G..............5'
 15

RESTRICTION MAPPING

Generally, a number of different strategies are required to construct detailed accurate

restriction enzyme maps of DNA. The plasmid used in this lab is relatively small, circular,
contains a multicloning site and has few sites cut by restriction enzymes. It will be sufficient to
use the technique of single digests of the plasmid DNA to map restriction sites that cut once into
the insert and once in the multicloning site. If a restriction enzyme is not included in the
multicloning site or cuts more than once in the insert then multiple restriction enzyme digestion
of plasmid DNA is required. Other techniques that are commonly used are: (1) sequential
digestion of an isolated DNA fragment with a second restriction enzyme, (2) partial digestion of
unlabelled or one terminal end labelled, and (3) partial exonucleolytic DNA digestion followed
by digestion with restriction enzyme.

AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is the standard method to separate and identify DNA
fragments. The technique quickly and easily resolves DNA fragments. Location of DNA
fragment bands can be immediately detected by staining with the fluorescent, intercalating dye
ethidium bromide.

The electrophoretic migration rate of DNA through agarose is dependent on molecular

size of the DNA, agarose concentration, conformation of the DNA, and applied current. Base
composition and temperature have very little effect. Linear double stranded DNA migrate at a
rate that is inversely proportional to the log of their molecular weights. Agarose concentrations
of 0.3% to 2.0% permit separation of linear DNA molecules from 0.1 kb to 60 kb. Closed
circular, nicked circular, and linear DNA of the same molecular weight migrate through agarose
gels at different rates. Closed supercoiled circular DNA generally migrates the fastest. Other
forms that may be present are linear, nicked circular and dimer, trimer, concatenate forms of
superhelical DNA. The relative mobilities are influenced by agarose gel concentration, strength
of applied current, ionic strength of buffer, density of superhelical twists, and concentration of
ethidium bromide. When considering the applied current, the rate of linear DNA migration is
linear to the applied current at low voltage. As the electrical field strength is increased, high
molecular weight DNA mobility is increased differentially resulting in effective range of
separation decreasing as voltage increases. Generally maximum resolution is obtained at 5
V/cm.
 16

ISOLATION OF PLASMID DNA

All procedures of plasmid DNA isolation involve (1) growth of bacteria and
amplification of plasmid, (2) harvesting and lysis of bacteria, and (3) purification of plasmid
DNA. Normally there are ten to two hundred plasmids (relaxed - not connected to chromosomal
replication) per bacterial cell. The bacteria containing the plasmid are grown in LB medium plus
antibiotic that selects for plasmid and may or may not be amplified by the addition of
chloramphenicol late in log phase. The chloramphenicol selectively inhibits chromosomal DNA
synthesis and amplifies plasmid DNA synthesis.

The isolation procedures takes advantage of major differences between genomic DNA
and plasmid DNA. By gentle bacterial lysis small molecules, including covalently closed
supercoiled plasmids are released into solution. Large molecules such as chromosomal DNA
fragments are trapped in the cell debris.

The cells can be lysed by boiling, alkali, detergents such as sodium dodecyl sulphate (SDS)
or Triton and phenol. Each method is appropriate for different experimental expectations. Lysis
by non-ionic detergent (Triton) is considered the most gentle and is especially good for isolation
of plasmids over 10 kb. Heating or mild alkali (up to pH 12.5) treatment breaks most of the
hydrogen bonds in DNA. Closed circular plasmids regain their native configuration when
slowly cooled or returned to neutral pH while chromosomal DNA fragments remain in the
denature state. Isolation of plasmid DNA by rapid phenol precipitation produces the least pure
plasmid preparation (can not be digested with restriction enzyme).

The plasmid DNA can be purified by ethanol precipitation, ultracentrifugation in CsCl, and
application to silica or resin column.

HYBRIDIZATION

Hybridization is the formation of double stranded nucleic acid molecules by homologous

complementary strands. Generally fixed single stranded nucleic acid (solid supports, southern
blot or plaque blot) are screened with homologous single stranded radioactive probe.
Hybridization parameters are varied depending on the degree of homology between the
'homologous or near homologous' strands of nucleic acid. The formation of nucleic acid hybrids
is a reversible process. The melting temperature (Tm) of double stranded DNA is defined as the
temperature when half the duplex molecules have dissociated. The Tm is affected by monovalent
cation concentration (molar), base composition, length of nucleotides in shortest single strand,
and concentration of helix destabilizing agents. The stability of duplex formation between
strands with mismatched bases is decreased according to the number and location of mismatches.
For duplex formation between single strands with high homology the stringency of the
hybridization should be high to maintain a low background by adjusting the temperature and salt
concentration. There is approximately 16oC increase in Tm for each log increase in cation
concentration. Hybridization stringency can be altered during hybridization or washing. For
duplex formation with reduced homology the stringency of either the hybridization or the wash
must be decreased by lowering the temperature or increasing the salt concentration. However,
the wash must be stringent enough to allow differentiation between the positvie lamba clone -
probe binding and nonspecific background binding.
 17

CHEMILUMINESCENCE DIG HYBRIDIZATION

(Roche Molecular Biochemicals DIG application manual)

Random Primed Labeling of DNA

As the name implies the DNA probes is randomly label. The probes is linearized with a
restriction enzyme and denatured to single stranded DNA by boiling. Random hexanucleotides
primers that have the capability to attach to any part of the DNA are annealed to the DNA probe.
These hexanucleotides serve as primers for DNA synthesis (5' to 3') by the Klenow fragment of
DNA polymerase I of E. coli. Klenow polymerase, which lacks 5' to 3' exonuclease activity of
DNA polymerase 1, synthesis DNA incorporating the nucleotides plus some dUTP nucleotides
that are labelled with a molecule of digoxygenin (DIG) conjugated to alkaline phosphatase (AP).
UTP-DIG-AP is incorporated approximately every 10 nucleotides.
Hybridization
The probe is specific for the calmodulin gene, and under moderately stringent hybridization
conditions, hybridizes only with plaques in the Achlya EMBL3 library that contain calmodulin
DNA.
Chemiluminescent Detection
Once bound to the filter, the probe is detected by an immunological method that utilizes an
antibody (rabbit IgG) specific for the digoxygenin molecule that is linked to alkaline
phosphatase (figure 5). The chemiluminescent alkaline phosphate substrate, CSPD detects the
probe-target DNA hybrid. The conjugated alkaline phosphatase reacts with CSPD substrate
generating light. The light is detected with X-ray film.
 18

LIGATION REACTION

T4 DNA ligase catalyses the formation of phosphodiester bonds between adjacent 3'-OH
and 5'-P termini in double stranded DNA that has either cohesive ends or blunt ends. T4 ligase
requires both magnesium and ATP. Optimal conditions for DNA ligation are outlined in
FOCUS vol. 8 No. 1 (Winter 1986).

TRANSFORMATION OF E. COLI BY PLASMID DNA

Mandel and Higa (1970) first demonstrated that the uptake of lambda DNA by E. coli is
enhanced by treatment of E. coli cells with calcium chloride under cold conditions. Many
techniques now exist for preparation of competent cells, for example Hanahan (1983); all
directed towards optimizing the efficiency of transformation. Even under optimum conditions,
only a small portion of the bacterial cells are competent for a short period of time. Generally 1
DNA molecule in 10000 is successful at transformation but this depends largely on the E. coli
strain. New strains have been constructed that have a very high efficiency of transformation.

Once the plasmid DNA is inside the bacteria, the plasmid DNA replicates and expresses
the drug-resistance marker that allows the transformed cell to survive in the presence of
antibiotic. In this experiment, pM13 carries the drug resistant gene for ampicillin which is added
to LB plates for selection of transformants.

Recombinant plasmid transformants require a second marker gene. In this experiment,

pM13 $-gal is inactivated by inserted genomic DNA (recombinant) and when grown on plates
containing both ampicillin and X-gal/IPTG, white colonies are recombinants while blue colonies
are only transformants. White colonies cannot metabolize X-gal in the presence of IPTG. If X-
gal is metabolized (active Lac Z gene) blue colonies result.
 19

PROCEDURES
Note: Always hold all enzymes used in the recombinant DNA lab by the top of the tube
away from the enzyme.

LAB 1 SCREENING OF A PHAGE GENOMIC LIBRARY FOR A

PARTICULAR GENE

Week 1

Part I: Titration of Genomic Library

(Achlya library in EMBL3 arms)

1. A single colony of E. coli P2392, picked from a freshly streaked LB plate, was grown
overnight in 10 ml LB broth containing 10 mM MgSO4 at 39oC with shaking.

2. Prepare a 10-fold serial dilution to 10-9 of Achlya EMBL3 lambda genomic library. The
original Achlya 8 EMBL3 library is stored in DMSO at -70oC. Library used for lab is an
amplified library stored at 4oC over chloroform.
Preparation of 10 fold- serial dilutions: Prepare 10-1 dilution in a total volume of 200 :l
and dilutions 10-2 to 10-9 in a total volume of 1 ml. Start serial dilution by adding 20 :l of
amplified library to 180 :l saline (10-1), vortex. Next transfer 100 :l of 10-1 dilution to
0.9 ml saline (10-2 dilution), vortex. Then transfer 100 :l of 10-2 dilution to 0.9 ml saline
(10-3 dilution), vortex - repeat this process until you have prepared up to 10-9 dilution.

3. Plate 10-5 to 10-9 dilutions in duplicate. For these dilutions, set up sterile tubes
(duplicate) containing 0.2 ml plating bacteria and 0.1 ml library dilution. Also set up
one tube containing only 0.2 ml plating bacteria, negative control that demonstrates the
appearance of a lawn of bacteria.

5. Incubate without shaking at 39oC for 20 min. Selective for EMBL3 8 arms containing
genomic insert.

6. Quickly add 3.5 ml melted (55-60oC) 0.7% top LB-Mg agarose to one tube. If tube is too
hot, cool slightly before adding, but do not allow to start to solidify. Immediately pour
onto a labelled prewarmed (39oC) LB plate and swirl the plate gently to ensure an even
distribution of bacteria and top agar. Repeat with each of the tubes.

7. Let the plates stand at room temperature for 5 min to allow the top agar to harden. Invert
the plates and incubate at 39oC overnight. Count number of plaques on all countable
plates.

8. Determine lambda library titer (plaques/ml) and volume required to add 10,000 phage
before coming to lab next week.
 20

Week 2

Part II: Preparation of Genomic Library Plaque Blots

1. A single colony of E. coli P2392, picked from a freshly streaked LB plate, was grown
overnight in 10 ml LB broth containing 10 mM MgSO4 at 39oC with shaking.

2. Set up in duplicate, adsorption tubes containing 0.3 ml plating bacteria (E. coli P2392)
and 10,000 phage from Achlya 8 EMBL3 library (volume determined from titration
results of Part I of this lab). The volume of phage library should not exceed 0.3 ml.

3. Incubate without shaking at 39oC for 20 min.

4. Quickly add 8 ml melted 0.7% top LB-Mg agarose and immediately pour onto a 150 mm
LB agar plate (prewarmed at 39oC). The agarose MUST BE COMPLETELY
DISSOLVED to ensure smooth spreading of top agar. Cannot be used for blotting unless
smooth surface.
Note: The plates must be dry, otherwise the layer of top agarose will peel off with the
filter.

5. Allow to solidify, incubate overnight at 39oC.

Next Day

6. Put all plates at 4oC for minimum of 2 hours. This helps prevent the top agarose from
peeling off when removing plaque filter blot. Select one plate for blotting.

7. Use specially designed nylon membranes for chemiluminescent detection - microporous,

hybdrophilic, neutral nylon. Carefully label nylon plaque blot with your GROUP
NUMBER, names and date. Labeling must be clear as demonstrators will carry out the
hybridization of your blots. Press hard enough to ensure marking indentation as pencil
itself will most likely wash off when hybridizing. Carefully place membrane on the
agarose surface. This is done by holding the filter on two sides with gloved hands or
forceps curving the filter upwards on the sides. Lower the filter onto the surface of the
plate touching the centre section first, then lowering the edges as the filter wets. Using a
sterile needle, mark the edges of the filter in 3 places (asymmetrically) by piercing the
needle through the filter into the agar. This ensures correct orientation of plaques.
Note: After removing filter and replacing petri plate lid, turn plate over and mark pierced
agar position with a felt pen.

8. Remove filter after 2 minute and carefully place, colony side up, on 3MM paper soaked
in denaturing solution. Do not submerge but float on surface. Leave for 5 min ONLY as
longer time will lyse the bacterial lawn around phage plaques causing high background.

9. Place filter, colony side up, on a 3MM paper soaked in neutralizing solution. Leave for 3
minutes then repeat with a fresh 3MM soaked in neutralizing solution.

10. Wash filter in 2x SSC buffer. Submerge, leave 1 min, then remove to air dry on 1MM
paper colony side up. Cover filter with saran wrap and expose to UV transilluminator for
2 min (colony side next to transilluminator). Attach a piece of masking tape to the saran
wrap labelled with your group # (most important), names, and date.

11. Store blot under vacuum at room temperature until ready to use or prehybridize
immediately.
 21

Week 3

Part III Chemiluminenscent DIG-labeled Hybridization (1)

DIG-High Prime DNA Labeling and Detection Kit II (cat. no. 1 585 614) contains
• DIG-High Prime labeling mix (5x conc.) - vial 1
• DIG-labeled control DNA - vial 2
• DNA dilution buffer - vial 3
• anti-DIG-alkaline phosphatase conjugate - vial 4
• CSPD chemiluminescent substrate (ready to use) - vial 5
• blocking solution (10x conc) - vial 6
• DIG easy-Hyb granules - vial 7

Parts A, B, C & D are done by the TA. However, students are responsible for all parts of the
experiment for lab exam.

A. Random primed DNA labeling of probe

1. Plasmid pCM116 (2 :g), which contains eel calmodulin cDNA, is linearized by

restriction enzyme digestion with Pst1 or BamH1.

2. Boil for 10 min to denature plasmid (single stranded DNA). Immediately put in
ice/water.

3. Add the following components in order to an eppendorf tube on ice.

• 10 ng-3 µg ice cold denatured linearized plasmid
• add sterile double distilled water to 16 :l
• 4 :l DIG-High Prime (contains hexanucleotide primers, dNTPs, dUTP-DIG-AP,
Klenow and appropriate salts) - mix thoroughly before using

4. Incubate at 37oC for a minimum of 1 hour.

5. Add 2 :l 200 mM EDTA, pH 8.0 to stop the reaction.

B. Determination of Probe Yield by Direct Detection Procedure

Require to ensure that you add the correct amount of probe to the hybridization.

1. Serial dilution of probe: Add 2 :l DIG-labeled probe to 38 (1/20) DNA dilution buffer.
Mix and transfer 5 :l to 45 :l (1/10 x 1/20) DNA dilution buffer. (Repeat 1/10 dilution
five more times.
2. Serially dilute DIG-labeled control DNA (stock is 5 ng/:l - dilute to 1 ng/:l) as step 1
starting with 1 ng/:l stock.
3. Using positively charged nylon filter (3 x 5 cm), apply 1 :l spots of each dilution of
probe in a row. Directly below and in the same order apply 1 :l spots of each control
DNA dilution. Mark location of each spot with a pencil.
4. Crosslink DNA to blot by placing a UV transilluminator for 3 min.
5. Place blot in 20 ml wash buffer (shake stock solution before adding) in a small plastic
container (top of tip box). Incubate for 2 min with shaking on flat rotary shaker. Discard
the wash buffer.
6. Add 10 ml blocking solution. Incubate for 30 min. Pour off and discard solution.
7. Add 10 ml antibody solution. Incubate for 10 min. Pour off.
8. Wash twice (2x 15 min) with 10 ml amounts of wash buffer.
9. Equilibrate for 5 min in 10 ml detection buffer.
10. Place blot between two acetate sheets.
 22

11. Apply 0.1 ml (about 4 drops) of ready-to-use CSPD alkaline phosphatase substrate
quickly across the surface of the membrane (do not allow the membrane to dry). As you
apply, cover area with upper inner side of plastic bag - flip upper sheet up and down to
spread the substrate. Cover smoothly - no air bubbles.
12. Incubate for 5 min at room temperature.
13. If excess liquid present, squeeze out.
14. Place acetate covered blot in sealable container on moistened paper towels. Seal lid.
Incubate membrane for 15 min at 37oC. This step is required for activation of CSPD.
15. Expose the blot inside the clear acetate to X-ray film for 30 min. Develop film. If no
highlighted plaques are apparent. Expose to film for 1 or 2 days. Develop film.
16. Compare dot intensity of your probe to control DNA. An excellent concentration is 20
ng/:l. For best results your DIG-labelled probe should have similar intensity to control
DNA.

Solution components:
maleic acid buffer: 0.1 M maleic acid, 0.15 M NaCl, adjust to pH 7.5 with solid NaOH.
wash buffer (maleic acid buffer plus tween20): 0.1 M maleic acid, 0.15 M NaCl, pH 7.5, 0.3% (v/v) Tween 20
blocking solution: dilute blocking solution (purified fraction of milk powder) 1:10 with maleic acid buffer
antibody solution: Anti-DIG-alkaline phosphatase conjugate vial 5 - centrifuge for 5 min at 10000 rpm in original
vial. Remove an aliquot and dilute 1:10000 with blocking solution.
detection buffer: 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl

C. Prehybridization and Hybridization of plaque blot

1. Place one blot in a hybridization roller tube. Add 10 ml standard hybridization solution
(5x SSC*, 1.0% (w/v) Blocking Reagent for nucleic acid hybridization, 0.1% N-
lauoylsarcosine, 0.02% sodium dodecyl sulfate (SDS). Prehybridize for at least 1 hour at
65oC.
*
5x SSC: 750 mM NaCl, 75 mM sodium citrate, pH 7.0.

2. Place the required amount of DIG-labelled probe in an eppendorf tube (10-25 ng/ml).
Add 50 :l sterile double distilled water. Heat for 5 min in boiling waterbath.
Immediately place of ice.

3. Remove 5 ml of the prehybridization solution. Add DIG-labeled probe to remaining 5

ml of hybridization solution in roller tube containing plaque blot.

4. Hybridize overnight at 58oC in roller tubes.

D. Plaque blot washing

1. Pour hybridization solution into container. Freeze at -20oC or immediately reuse as may
be used many times. Add 20 ml 2x SSC + 0.1% SDS to hybridization bottle containing
blot. First shake to wash in behind blot then put on roller. Wash for 5 min in at room
temperature. Discard wash solution. Repeat wash. This wash removes unbound probe.

2. Add 20 ml 0.1x SSC containing 0.1% SDS (pre-warmed to 58oC). Wash the blot on the
roller for 15 min at 58oC. Discard wash solution.
 23

STUDENT LAB STARTS HERE - may change as directed in pre-lab

E. Chemiluminescent Detection of probe-plaque hybrid

It is important that the blot DOES NOT DRY during any of the following steps.
See Part II for solution components.

1. Place blot in 40 ml wash buffer (shake stock solution before adding) in a small plastic
container (top of tip box). Incubate for 2 min with shaking on flat rotary shaker. Discard
the wash buffer.
2. Add 40 ml blocking solution. Incubate for 30 min. Pour off and discard solution.
3. Add 15 ml antibody solution. Incubate for 30 min. Pour off.
4. Wash twice (2x 15 min) with 40 ml amounts of wash buffer.
5. Equilibrate for 5 min in 20 ml detection buffer.
6. Place blot between two acetate sheets.
7. Apply 600 :l CSPD alkaline phosphatase substrate across the surface of the membrane (do
not allow the membrane to dry). As you apply, cover area with upper inner side of clear
acetate sheet - flip upper sheet up and down to spread the substrate. Cover smoothly, no air
bubbles.
8. Incubate for 5 min.
9. If excess liquid present, squeeze out.
10. Place acetate covered blot in sealable container on moistened paper towels. Seal lid.
Incubate membrane for 15 min at 37oC. This step is required for activation of CSPD.
11. Expose the blot inside the clear acetate to X-ray film for 30 min. Develop film. If no
highlighted plaques are apparent. Expose to film for 1 or 2 days. Develop film.

Part IV: Selection of Positive Clones

1. Positive clones are located by plaques that 'light up'. Align X-ray film with blot and mark
orientation positions. Using the orientation marks on plate align with autoradiogram.
Mark plaques that align with 'light up' spots.

2. For each positive spot set up a sterile eppendorf tube containing 0.5 ml phage buffer and 3
drops of chloroform.

3. Pick plaques by using a Pasteur pipette equipped with a rubber bulb, stab through the
chosen plaque into the hard agar beneath. Apply mild suction so that the plaque, together
with the underlying agar, is drawn into the pipette.

4. Eject the fragment of agar containing the positive plaque into the phage buffer. Add a few
drops of chloroform. Store positive phage clone at 4oC. An average plaque yields 105 to
106 phage particles.
Comment: If the plaques are not well separated it is necessary to repeat the screening
process (plating, plaque hybridization ) to ensure working with a single clone. In this case,
need to use only 90 mm petri plates and screen 50 - 150 plaques.
 24

LAB REPORT

Data Presentation and Data Analysis

Part I: Genomic Library Titration: Achlya-EMBL3

1. Tabulate titration results.

2. a) Determine titre (pfu/ml).

b) Determine volume required per plate that represents an entire library (10,000 plaques).
Dilutions must be included in answer if volume is smaller than can be measured using a
pipetman.

Part III: Plaque Hybridization

1. a) Include a completely labelled figure of the plaque blot.

b) How many clones do you expect to “light up”(hybridize) per plate? Explain.

Questions:

1. The Achlya genomic DNA library used in your lab has been amplified many times. What
problem is associated with an amplified library? Suggest a possible solution.

2. Outline how genomic DNA is prepared prior to insertion into lambda EMBL3 for
preparation of a library. Explain each major step.

3. Explain how to determine the number of EMBL3 8 plaques required to represent one
genome of Achlya. Numerical calculations are required.
Achlya genomic size is ~ 45 x 103 kb.

4. What experimental steps optimize hybridization and ensure stringency (low background)?

5. Summit a question about any part of this experiment that you do not understand. Questions
will be collected and discussed during Lab exam question period, week 11.
Notes:
(1) For this lab, significant lambda phage plaque count numbers are between 30 and as high as plaques can be
counted accurately (not overlapping). If no phage plaque counts are above 30 use numbers below 30 to
calculate the number of pfu/ ml - state below significance but only data available. pfu = plaque forming units
(2) For each phage stock culture there should only be one calculated pfu/ml value, that is, average duplicate
plates, then average dilutions if more than one dilution in significant plate count range.
(3) Footnote values in table that are used to calculate titre.
 25

LAB 2 SUBCLONING OF GENOMIC DNA FRAGMENT INTO pM13 PLASMID

Week 3

Part I: Restriction enzyme digestion of genomic library clone and purification

(A) Preparative restriction enzyme digestion of genomic library clone

Note: To ensure that enough genomic inserted is isolated by the class, some groups will use the
pM13cam7Cla2.1 plasmid DNA (contains Achlya Cla1 2.1 kb fragment same as cam7 8 EMBL3
clone) to isolate Cla1 2.1 kb DNA fragment. The DNA sample used by each group will be
assigned in pre-lab.

1. Restriction enzyme reaction:

addition amount added (volume = 70 :l)
_____________________________________________________________
library clone ( :g/:l)
(cam7-EMBL3 8 DNA) 10 :g
OR pM13cam7Cla2.1DNA 5 :g

Reaction buffer #1 7 :l
distilled water* to 70 :l
Cla I (10 U/:l) 2 U/:g DNA

* do not discard distilled but return to supply bench as used later in the term.

Please read restriction enzyme digestion comments prior to setting up reaction tubes:
a. Always keep restriction enzyme on ice and return to -20oC as quickly as possible.
b. Always handle restriction enzymes and all biological materials by the cap region of
the tube. Warm hands may destroy or shorten the lifetime of the enzyme.
c. Each restriction enzyme has a corresponding specific reaction buffer.
d. Add components using pipetman by touching the inside of the eppendorf tube about
0.5 cm below top of tube and releasing solution. A good idea is to place
components at different spots around the inside of the tube.
e. Add restriction enzyme last.
f. Restriction enzyme: 1 unit of restriction enzyme is the amount of enzyme required
to completely digest 1 :g of bacteriophage lambda DNA in 1 hour at 37oC.
Restriction enzyme may need to be added at a concentration of 1-8 units/:g
plasmid DNA.
g. Mix components together to start digestion by spinning in a microfuge for 2-5 sec.
This is a general procedure for mixing all small volumes.

2. Incubate at 37oC for 1.5 h. Stop reaction by adding 14 :l agarose stop solution.

3. Store sample at -20oC until next week. Clearly label tube with group number and initials.
A demonstrator will prepare and load the gel as outlined in the following step 4 the
morning of the lab.
 26

Week 4

[Step 4 - is done by the

demonstrator.] agarose gel comb

4. Use BIO-RAD DNA

sub-cell
electrophoretic unit
and 240 ml 0.7% Thin tape covering three wells inclusive.
agarose prepared in 1x
TAE buffer. Add Figure 5. Well comb for preparative agarose gel
ethidium bromide just electrophoresis.
before pouring (7
:l/200 ml agarose gel). If using the large gel units, add 280 to 300 ml agarose to gel tray. The
well comb is taped to enable loading of a large sample (Figure 5). Using plastic tape - tape off 2
wells (inclusive) and prepare agarose gel as usual. Load as much sample as possible in enlarged
well and use 1x TAE as the running buffer. Load 7 :l 1 Kb Plus Ladder at each end (single well).
Do not use TBE (Tris-borate buffer) which interacts with agarose and reduces the recovery of
DNA from the gel. Note: Often when double stranded DNA fragments are isolated from agarose
gel they are stained for a short period of time after electrophoresis of gel
to reduce the time that ethidium bromide is in contact with the DNA. This reduces nicks in
the double stranded DNA caused by ethidium bromide in the presence of light. Also when
cutting the 2.1 kb DNA band out of the gel, the time should be as short a time as possible.
Again the reason is to reduce the nicks in double stranded DNA caused by ethidium
bromide in the presence of UV light.

5. Run at 80-100 volts for 4 hours.

6. Wear disposable gloves. Before handling samples rinse gloves with water to remove any
external powder. The powder may interfere with your experiment. Cut out band and place
in eppendorf tube keeping the size of the band as small as possible. Coarsely break gel up
using a metal spatula. Centrifuge the sample for several seconds to bring down the gel
slice. Estimate gel volume using graduated markings on the side of most eppendorf tubes.

(B) Purification of genomic restriction enzyme fragment

1. Add approximately 3 volumes Prep-A-Gene purification binding buffer (sodium

perchlorate) to the gel slice and agitate gently to dissolve. If necessary heat the tube at
55oC for 2 min. Calculate the 3 volumes Prep-A-Gene purification binding buffer required
using the volume of agarose gel.

2. Add 10 :l of Prep-A-Gene matrix per :g of DNA. Mix gently and incubate for 10 min
at room temperature. During this incubation time, mix the tube frequently by end over end
rotation.

3. Microfuge for 30 sec. Aspirate or remove supernatant with pipetman.

4. Rinse Step: Add 250 :l DNA purification kit binding buffer. Gently mix completely by
briefly vortexing several times.

5. Microfuge for 30 sec to pellet the matrix. Aspirate or remove supernatant with pipetman.
See TIP step 1 of plasmid DNA prep.
 27

6. Wash pellet twice with a 250 :l complete wash buffer (1:1 wash buffer:95% EtOH +
salts). Wash means to add wash buffer, do not mix or vortex, microfuge 30 sec and
aspirate off supernatant. Again add 250 :l wash buffer, microfuge for 30 sec and aspirate
off the supernatant completely. Do not disturb pellet, but it is important that all supernatant
is completely removed.

7. Resuspend Prep-A-Gene matrix pellet in 10 :l elution buffer by vortexing. Incubate at

50oC for 5 min. Microfuge for 1 min. Transfer supernatant to clean tube. Make sure no
Prep-A-Gene matrix pellet is removed but if this happens microfuge again microfuge and
transfer supernatant to another clean tube.

8. Clearly lab tube with group number and description. Store at -20oC until ready for agarose
gel electrophoresis and ligation experiment.

Solution components:
Binding Buffer: 6 M sodium perchlorate; 50 mM Tris-HCl (pH 8.0); 10 mM EDTA (pH 8.0)
Wash Buffer + EtOH: 400 mM NaCl; 20 mM Tris-HCl (pH 7.5); 2 mM EDTA (pH 7.5); 50% EtOH (v/v)

Part II: Preparation of pM13 plasmid DNA by Concert™ Rapid Plasmid Purification
System
Extracted from Life Technologies product information (GIBCO BRL Products)

Remember to use the P1000 pipettor for volumes between 200 and 1000 :l.
Remember to label all your tubes as you will also be isolating pM13cam7Cla2.1 at the same time.
Make sure TE buffer is preheated to 65oC - 70oC.
Each group is supplied with a fresh overnight 5 ml LB-AMP culture of E. coli JM109 containing
pM13.

1. Cell Harvesting: Put 3 ml bacteria culture of E. coli JM109 containing pM13cam7Cla2.1

into two eppendorf tubes (each tube only hold 1.5 ml). Centrifuge at room temperature for
1 min (12,000 x g). Remove supernatant by aspiration. Aspiration must be used to
completely remove all supernatant.
*TIP. When centrifuging any liquid where you are collecting a pellet in an eppendorf tube
alway place the tube in the microfuge with lid joiner facing outwards. When you remove
your tube from the microfuge you know where to look for the pellet; at the botton of the
tube below lid joiner. This allows you to keep the aspirator tip away from the pellet area if
the pellet is poorly visible.
2. Condensing two tubes to one tube and Cell Suspension: Add 250 :l Cell Suspension
Buffer (G1) to ONLY one pellet tube and completely resuspending the cells using the
pipetman. Transfer the resuspended pellet to the second pellet tube and again completely
suspend cells. The solution must be homogeneous or very little plasmid will be extracted.
3. Cell Lysis: Add 250 :l Cell Lysis Solution (G2) and immediately mix gently by inverting
the tube exactly five times. Do not vortex. Incubate at room temperature for 5 min. Do
not incubate longer than 5 minutes.
4. Neutralization: Add 350 :l Neutralization Buffer (M3) and mix immediately by inverting
the tube five times. Do not vortex. Centrifgue for 10 min (12,000 x g) at room
temperature.
5. Cartridge loading: Place a vacuum cartridge in a 2 ml wash tube (straight sided plastic
tube). Label tube and cartridge. Decant the supernatant into the cartridge. Decant by
quickly tipping your tube over the cartridge with top edges touching. Do not remove
remainder of supernatant with pipetman. It is important that none of the precipitate is
transferred to the cartridge. Centrifuge for 1 min ( 12,000 x g). Discard the flow-through.
 28

Cartridge contains silica based membranes that selectively binds plasmid DNA.
6. First Wash: Place the cartridge back in the 2 ml wash tube. Add 500 :l Optional Wash
Buffer (GX) to the cartridge. Incubate at room temperature for 1 min. Centrifuge for 1
min. Discard the flow-through. This step is required for nuclease rich bacteria.
7. Second Wash: Place the cartridge back in the 2 ml wash tube. Add 700 :l Wash Buffer
(G4) to the cartridge. Centrifuge for 1 min. Discard the flow-through. Centrifuge again
for 1 min to remove residual wash buffer.

8. Plasmid Elution: First cut off the cap of the 1.5 ml recovery tube. Then place the
cartridge into the 1.5 ml recovery tube. Add 75 :l warm (65-70oC) TE buffer directly to
the center of the cartridge. Incubate at room temperature for 1 min. Centrifuge for 1 min.
Discard cartridge and cap tube. This is your plasmid DNA prep. Clearly label tube with
plasmid name, DNA, concentration, group #, and your initials. After removing 5 :l to a
new labelled eppendorf tube for the spectrophotometer reading, store sample at -20oC in
student sample box. Do not mix up your samples as you are isolating two different DNA
plasmid preps.

9. Add 1 ml TE buffer* to your 5 :l plasmid DNA sample. Measure absorbance at 260 nm to

determine concentration of DNA.
*do not discard TE buffer, return to supply bench.
Cell Suspension Buffer (G1): 150 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 :g/ml RNase A
TE Buffer (TE): 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
Cell Lysis Solution (G2): 0.2 M NaOH, 1% SDS
Wash Buffer (G4): 200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA...dilute 1:1 with 95% EtOH
Optional Wash Buffer (GX): product proprietary formulation (contains acetate, guanidine hydrochloride, EDTA and ethanol)
Neutralizing Buffer (M3) product proprietary formulation (contains acetate and guanidine hydrochloride)

10. Calculate the concentration of your plasmid DNA sample before coming to lab next week.
Also calculate the volume required to add 0.5 :g pM13 plasmid DNA to your restriction
enzyme digest. Volume should not exceed 7 :l. Borrow plasmid DNA if your require a
greater volume than 7 :l.
Notes:
(i) An absorbance reading of 1 corresponds to approximately 50 :g/ml for double stranded
DNA. Maximum yield is 30 :g/cartridge.
(ii) The yield of plasmid DNA is dependent on the plasmid copy number, plasmid type,
bacterial strain, and growth conditions.
 29

Week 5

Part III: (A) pM13 plasmid restriction enzyme digestion

Note: All spins in the microfuge are done at room temperature unless otherwise stated.
1. Set up the following restriction digestion in an eppendorf tube:
addition amount added
___________________________________________
pM13 DNA(* :g/:l) 0.5 :g
Reaction Buffer #1 1.5 :l
distilled water to 15 :l
Cla I (10 U/:l) 1 :l

* Determine volume to add from your DNA concentration before coming to lab. If unsure
of your calculation, check with the lab demonstrator. Do not exceed 7 :l pM13 plasmid
DNA added to your reaction. Borrow plasmid DNA if required.

2. Incubate at 37oC for 1 h.

3. Stop reaction by heating at 65oC for 20 min. Store at -20oC until ready for ligation.

Part III: (B) Agarose Gel Electrophoresis of pM13 (RE digested and uncut) and
genomic insert

1. Set up 0.7% agarose gel.

2. Load the following samples . Make sure you record where and what order you loaded your
samples on the demonstrator agarose gel diagram.
Standard samples:
5-7 :l 1 kb Ladder Plus standard (50 ng/:l) in both end wells (DNA sample do not always
run at 90o angle to well line therefore it is important to use the two end wells for standard)
Student samples- all groups must load in the same order, 1 through 4:
(1) 4 :l Cla digested pM13 + 2 :l stop solution + 6 :l distilled water
(2) 4 :l uncut pM13 (your own pM13 preparation even if less than 0.1 :g/:l) + 2 :l stop
+ 6 :l distilled water
(3) 4 :l Prep-A-Gene Cla1 2.1 kb insert + 2 :l stop solution + 4 :l distilled water
(4) 4 :l uncut pM13cam7Cla2.1 (your own pM13cam7Cla2.1 preparation even if less than
0.1 :g/:l) + 2 :l stop solution + 6 :l distilled water

3. Electrophoresis overnight at 15 volts or 3 hours at 80 to 100 volts.

4. Record gel data. Step 3 and 4 are done by your demonstrators and a printout of you gel will
be returned to you next lab period. Data will also be posted on the website as soon as
possible after photographing.

Notes:
(i) Refer to appendix for agarose gel electrophoresis instructions.
(ii) Distilled water is added to samples to increase volume to make loading of sample easier.
 30

Week 7

5. Monday 9:00 am after mid-term break: Read the next part of the experiment before
lab day as you need to set up your experiment by 9:00 am.

Part IV: Ligation of plasmid and genomic fragment

NOTE: The ligation reaction must be set up first thing the morning of the lab (4 to 6 hours
incubation time at room temperature).
1. Set up the ligation reaction in an eppendorf tube as follows:

addition amount added

_____________________________________________________________
pM13 Cla I digest 1 :l
genomic DNA fragment 4 :l
[cam7Cla2.1 insert]
5x ligase buffer 3 :l
T4 ligase* 2 :l
distilled water to 15 :l
*remember to keep the T4 ligase on ice and return to freezer after using.

2. Incubate at room temperature for 4-6 hours.

Day 1

Part V: Transformation of E. coli and Selection of Recombinants

A. Preparation of competent E. coli JM107

1. Add 0.2 ml of an overnight culture of E. coli JM107 to 10 ml LB + magnesium medium.

Shake at 37oC for 2.5 h. Put on ice for 20 min.

STUDENT LAB STARTS HERE

2. Centrifuge at 10000 rpm for 5 min at 4oC using sterile plastic screw cap centrifuge tubes.
Decant supernatant and completely remove remaining supernatant by aspiration or
pipetman. Gently resuspend pellet in 0.5 ml 50 mM calcium chloride. Put on ice 15 min.
The bacteria should now be competent.

B. Transformation of competent JM107

1. In a sterile eppendorf tube mix 50 :l of TFB (transformation) buffer and 10 :l ligation

mixture. Add 100 :l of competent E. coli. Set up two other reactions; a negative control;
replace DNA with TFB, and a positive control; 10 :l pM13 DNA (0.1 :g/10 :l DNA). Let
the reaction tubes sit on ice for 15 min.

2. Transfer to 42oC waterbath for 2 min.

3. Add 0.8 ml of prewarmed LB + Mg++ (37oC) to the mixture and incubate in a 37oC
waterbath without shaking for 40 min.
 31

C. Transformant and Recombinant Selection (X-gal/IPTG)

1. Positive control, pM13.

a) Prepare all dilutions using a total volume of 1 ml saline in each dilution tube.
b) Spread plate 0.1 ml of undiluted mixture, and 10-1, 10-2, 10-3 and 10-4 dilutions on LB-
AMP plates (duplicate).
c) Spread plate 0.1 ml of 10-4, 10-5, 10-6, 10-7, 10-8, and 10-9 on LB plates in duplicate.
d) Allow the plates to dry upright at room temperature, invert, and incubate at 37oC
overnight.

1.
1. 2. 3.
2. 4. 5.
3.
6. 7.
4. 8. 9.
5. 10. 11.
control control
6. 12.

Figure 6. Pick plate layout.

e) Count the number of colonies on all plates possible.

2. Negative Control, no plasmid.

a) Spread plate 0.1 ml in duplicate on LB-AMP plates.
b) Incubate at 37oC.
c) Check for growth of bacteria.

3. Ligation mixture (pM13-Cla I fragment).

a) LB-AMP plates containing 40 :g/ml X-gal in NN dimethyl formamide and 24 ng/ml
IPTG are used to select for transformants and recombinants. Spread plate a portion of the
transformation mixture by aliquoting 50 :l, 100 :l and 200 :l of transformation mixture in
duplicate (total of 6 plates).
b) Incubate 24 hours at 37oC. Transfer plates to 4oC to enhance color production if
necessary.
c) Count the number of colonies (white and blue separately) on all plates possible.
d) Pick apparent white colonies (maximum 10 colonies) onto LB-AMP-X-gal/IPTG plates
and one blue colony to use as control, refer to figure 6. These colonies are possible
recombinants.
e) Incubate overnight at 37oC. Again transfer to 4oC to enhance color production if
necessary. Store plates at 4oC until Friday before scheduled lab period.
 32

Day 5

Part VI: Verification of recombinant by sizing uncut plasmid DNA (QIAprep) on

agarose gel

1. Inoculate three 5 ml LB-AMP broths with white colonies and inoculate one LB-AMP broth
with a blue colony. Put in designated tray in student cold box. The TA will put the cultures
on the 37oC rotator Sunday night as require overnight growth.
*If your group has more than 5 white colonies leave on bench by board for other groups
after you set up your cultures. If your group does not have enough white colonies borrow
from another groups or use plates put on bench by board.

Week 8
2. Prepare 0.7% agarose gel. Place combs in tandem, ie., two combs per gel. Take Lab 3
restriction mapping experiment into consideration when calculating the number of agarose
gels required.

3. Isolate Plasmid DNA using QIAGEN Spin Miniprep plasmid DNA Kit Protocol (1).
Repeat the following protocol for each culture

Cell Harvesting: Put 3 ml bacteria culture of E. coli JM109 containing pM13cam7Cla2.1

into two eppendorf tubes (each tube only holds 1.5 ml). Centrifuge at room temperature
for 1 min (12,000 x g). Remove supernatant by aspiration. Aspiration must be used to
completely remove all supernatant.
Condensing two tubes to one tube and Cell Suspension: Add 250 :l Buffer P1 to
ONLY one pellet tube and completely resuspending the cells using the pipetman.
Transfer the resuspended pellet to the second pellet tube and again completely suspend
cells. The solution must be homogeneous or very little plasmid will be extracted.
Cell Lysis: Add 250 :l Buffer P2 and mix gently by inverting the tube 4 -6 times. Do not
vortex. If necessary continue inverting until the solution becomes viscous and slightly
clear.
Neutralization: Add 350 :l Buffer N3 and mix immediately by inverting the tube gently 4
-6 times. Do not vortex. The mixture should become cloudy. Centrifgue for 10 min
(12,000 x g) at room temperature. A white pellet forms.
Cartridge loading: Place a cartridge in an eppendorf tube. Label tubes. Decant the
supernatant into the cartridge. Decant by quickly tipping your tube over the cartridge with
top edges touching. Do not remove remainder of supernatant with pipetman. It is
important that none of the precipitate is transferred to the cartridge. Centrifuge for 1 min (
12,000 x g). Discard the flow-through.
Optional First Wash: Place the cartridge in an eppendorf tube. Add 500 :l Buffer PB to
the cartridge. Centrifuge for 1 min. Discard the flow-through. This step is required for
nuclease rich bacteria.
Second Wash: Place the cartridge back in the 2 ml wash tube. Add 750 :l Buffer PB to
the cartridge. Centrifuge for 1 min. Discard the flow-through. Centrifuge again for 1 min
to remove residual wash buffer.
Plasmid Elution: First cut off the cap of the 1.5 ml recovery tube. Then place the
cartridge into the 1.5 ml recovery tube. Add 50 :l Buffer EB directly to the center of the
spin cartridge. Incubate at room temperature for 1 min. Centrifuge for 1 min. Discard
cartridge and cap tube. This is your plasmid DNA prep. Clearly label tube.

Note: you are not required to know the exact components of each buffer - however, they are almost
identical to the Concert™ plasmid protocol used elsewhere in this lab manual. Concert mini-
preps are no longer available from Invitrogen. This year is a transition year where I am using up
remainder of concert preps in department stock and switching to QIAGEN protocol.
 33

8. Electrophoresis for three hours at 80-100 volts.

9. Photograph the gel using the Gel Doc and save gel file.

10. A printout of you gel will be returned to you in class Thursday as Lab 2 report is due
Monday (Week 9). Data will also be posted on the website as soon as possible after
photographing.

LAB REPORT

Data Analysis

1. Calculate the concentration of pM13 and pM13cam7Cla2.1 DNA from absorbance reading
at 260 nm.

2. a) Include a completely labelled figure of printout of agarose gel for Part III (B). Also
include gel electrophoresis conditions in figure legend.
If you cannot analyze your own data, reference and use another’s groups expected data.

b) Using the 1 kb Plus standard visually estimate the size of Cla1 digested pM13. How
should your Cla1 digest of pM13 lane appear if it can be used for the ligation experiment?
Explain why.

c) What are the differences and similarities between your uncut pM13 and
pM13cam7Cla2.1 samples with reference to the different forms of plasmid DNA. Label
forms on figure 2a.

d) Do you have a visible Cla1 Prep-A-Gene fragment? How should your Cla1 Prep-A-
Gene sample lane appear before you can use the sample for the ligation reaction?

3. Transformation Results:
A. Negative Control
a) Record if growth/no growth of colonies.
b) Why include a negative control?

B. pM13 Transformation
a) Tabulate all data.
b) Determine the ability of E. coli to take up DNA. Express as ratio of number of
transformants to number of viable bacteria.
c) Determine the ratio of molecules that are successful at transformation. Molecular
weight of one bp = 650.

C. pM13-Cla I genomic fragment ligation mixture Transformation

a) Tabulate all data.
b) Calculate percentage of transformants that are recombinants.

4. a) Include a labelled figure of Quick Screen DNA Isolation Method agarose gel digital
camera printout.
b) Analyze your data. If your data is not as expected, reference another group’s expected
data and analyze. State conclusion.

Notes:
(i) For this lab, significant E. coli plate count numbers are between 30 and as high as bacteria can
be counted (colonies not overlapping). If no E. coli plate counts are above 30 use numbers below
 34

30 to calculate the number of bacteria per ml - state below significance but only data available.
However, if you have one white colony, that is significant because you are looking for
recombinants - usually one is all you need.
(ii) For each sample there should only be one calculated bacteria/ml value, that is, average
duplicate plates, then average dilutions if more than one dilution in significant plate count range.
(iii) Footnote values in table that are used to calculate titre.

Questions

1. In your lab 0.7% agarose was used to run your plasmid samples. Explain why 0.7% with
reference to the principle of agarose gel electroporesis. Possible percentage agarose gel
range is 0.3% to 2%.

2. According to Hanaham and Blooma (1966) there are three steps to plasmid DNA uptake by
bacteria. State the steps. What experimental procedures performed in your lab enhance
each step?
a
Hanahan, D. 1996 Mechanisms of DNA Transformation. In: Neidhardt FC, editor. Echerichia coli and
Salmonella typhimurium, 2nd ed., Washington: ASM Press. p 2449-2459.

3. Summit a question about any part of this experiment that you do not understand. Questions
will be collected and discussed during Lab exam question period, week 11.

References:
(1) QIAGEN Inc. 03/2002 In QIAprep Spin Miniprep Kit Protocol.
http://www.qiagen.com/literature/handbooks/qp/1019952_QPHB_prot01.pdf (accessed, June 13,
2002)
 35

LAB 3 RESTRICTION ENZYME MAPPING OF GENOMIC FRAGMENT

SUBCLONED IN pM13

Week 4

Part I: Preparation of pM13cam7Cla2.1 plasmid DNA by Concert™ Rapid Plasmid

Purification System
Extracted from Life Technologies product information (GIBCO BRL Products)

Remember to use the P1000 pipettor for volumes betweem 200 :l and 1000 :l.
Remember to carefully label all your tubes as you will also be isolating pM13 at the same time.
Make sure TE buffer is preheated to 65oC - 70oC.
Each group is supplied with a fresh overnight 5 ml LB-AMP culture of E. coli JM109 containing
pM13cam7Cla2.1.

CARRY OUT THE FOLLOWING PROCEDURE TWICE for E. coli JM109 pM13cam7Cla2.1

1. Cell Harvesting: Put 3 ml bacteria culture of E. coli JM109 containing pM13cam7Cla2.1

into two eppendorf tubes (each tube only hold 1.5 ml). Centrifuge at room temperature for
1 min (12,000 x g). Remove supernatant by aspiration. Aspiration must be used to
completely remove all supernatant.

2. Condensing two tubes to one tube and Cell Suspension: Add 250 :l Cell Suspension
Buffer (G1) to ONLY one pellet tube and completely resuspending the cells using the
pipetman. Transfer the resuspended pellet to the second pellet tube and again completely
suspend cells. The solution must be homogeneous or very little plasmid will be extracted.

3. Cell Lysis: Add 250 :l Cell Lysis Solution (G2) and mix gently by inverting the tube
exactly five times. Do not vortex. Incubate at room temperature for 5 min. Do not
incubate longer than 5 minutes.

4. Neutralization: Add 350 :l Neutralization Buffer (M3) and mix immediately by inverting
the tube five times. Do not vortex. Centrifgue for 10 min (12,000 x g) at room
temperature.

5. Cartridge loading: Place a cartridge in a 2 ml wash tube (straight sided plastic tube).
Label tubes. Decant the supernatant into the cartridge. Decant by quickly tipping your
tube over the cartridge with top edges touching. Do not remove remainder of supernatant
with pipetman. It is important that none of the precipitate is transferred to the cartridge.
Centrifuge for 1 min ( 12,000 x g). Discard the flow-through.
Cartridge contains silica based membranes that selectively binds plasmid DNA.

6. First Wash: Place the cartridge back in the 2 ml wash tube. Add 500 :l Optional Wash
Buffer (GX) to the cartridge. Incubate at room temperature for 1 min. Centrifuge for 1
min. Discard the flow-through. This step is required for nuclease rich bacteria.

7. Second Wash: Place the cartridge back in the 2 ml wash tube. Add 700 :l Wash Buffer
(G4) to the cartridge. Centrifuge for 1 min. Discard the flow-through. Centrifuge again
for 1 min to remove residual wash buffer.

8. Plasmid Elution: First cut off the cap of the 1.5 ml recovery tube. Then place the
cartridge into the 1.5 ml recovery tube. Add 75 :l warm (65-70oC) TE buffer directly to
the center of the spin cartridge. Incubate at room temperature for 1 min. Centrifuge for 1
min. Discard cartridge and cap tube. This is your plasmid DNA prep. Clearly label tube
 36

with plasmid name, DNA, concentration, group #, and your initials. After removing 5 :l
to a new labelled eppendorf tube for the spectrophotometer reading, store sample at -20oC
in student sample box.

9. Add 1 ml TE buffer* to your 5 :l plasmid DNA sample. Measure absorbance at 260 nm to

determine concentration of DNA. An O.D. reading of 1 corresponds to approximately 50
:g/ml for double stranded DNA. Maximum yield is 30 :g/cartridge. Note: The yield of
plasmid DNA is dependent on the plasmid copy number, plasmid type, bacterial strain, and
growth conditions.
*do not discard TE buffer, return to supply bench.

10. Calculate the concentration of your plasmid DNA sample before coming to lab next week.
Also calculate the volume required to add 0.5 :g pM13cam7Cla2.1 plasmid DNA to your
restriction enzyme digest. Volume should not exceed 7 :l. Borrow plasmid DNA if your
require a greater volume than 7 :l. An O.D. reading of 1 corresponds to approximately 50
:g/ml for double stranded DNA. Maximum yield is 30 :g/cartridge. Note: The yield of
plasmid DNA is dependent on the plasmid copy number, plasmid type, bacterial strain, and
growth conditions.

Week 8

Part II: Restriction enzyme mapping (single digests)

1. Set up six restriction enzyme reaction tubes as follows:

(a) 1.5 :l Reaction buffer (appropriate reaction buffer # for BRL enzymes)
(b) 0.5 :g* pM13+cam7Cla2.1 plasmid
(c) to 15 :l deionized water
(d) restriction enzyme as follows:

Tube # Restriction Amount added BRL reaction

enzyme buffer #
__________________________________________________________
1 Cla I 1 :l 1
2 Pst I 1 :l 2
3 Xba I 1 :l 2
4 Eco RI 1 :l 3
5 Hind III 1 :l 2
6 Sal 1 1 :l 10
*if absorbance reading seems to be low but you have a moderately intense band on the
agarose gel just add 4 :l of your pM13cam7Cla2.1 plasmid DNA per reaction. Do not
exceed 7 :l plasmid DNA per reaction tube.

2. Incubate in a 37oC waterbath for 1 hour. Prepare 0.7% agarose gel. Place combs in
tandem, ie., two combs per gel. Take Lab 2 Part VI. experiment into consideration when
calculating the number of agarose gels required.

3. Add 3 :l stop solution.

4. Load 7 :l 1 kb ladder plus standard in the end well of each row. All groups must load in
the same order, digests 1 through 6. Make sure you record where and what order you
loaded your samples on the demonstrator agarose gel diagram.

Step 5 & 6 are performed by your demonstrators and a photocopy or printout of you gel will be
returned to you next lab period. Data will be posted on the website as soon as possible after
 37

photographing.

5. Electrophoresis (0.7% agarose gel) using Tris-acetate buffer system at 80-100 volts for
approximately three hours.

6. Next day, place gel in BioRad Gel Doc 1000 unit (consists of enclosed box with camera
positioned on the top of the box. The gel is placed in the box over the UV transilluminator.
The box door is closed. The digital camera view is analysed by an attached computer using
the software package, Analyst Program. The file is saved, manipulated and printed.

LAB REPORT

Data analysis

1. Present a completely labelled figure of the Gel Doc printout of agarose gel (single digests).

2. Present a standard curve figure of log bp size (1 kb ladder plus standard fragments) vs
distance migrated. Include a table of data used for plot. Also include in table log bp size
values if not using semi-log graph paper. Semi-log paper may be used to plot standard
curve.

3. Present a table for all restriction enzyme fragment data.

(i) Using the standard curve determine the restriction digests fragment sizes (bp or kb).
Size may be determined by direct extrapolation on the graph or by using the slope
(standard graph paper only). Check agarose gel printout carefully as low bp size DNA
fragments may be faint.
(ii) Also include a column for total kb size for each gel lane.
(iii) As the total kb size is not always the same for each restriction enzyme digestion, all
size determinations should be normalized. This is done by multiply each digest by a
normalizing factor such that all fragments for each digest add up to the same value (~5.1
kb). Include a column of normalized fragment size values in the table.

4. Present a completely labelled figure of the restriction map. For the figure use normalized
data and orientate the genomic gene fragment with respect to the promoters, T3 and T7.
Draw map to scale on graph paper and label completely.
Note: When map is in kb, a difference of few bp is consider insignificant.
Is it possible to determine the location of restriction enzyme sites in the insert DNA if three
DNA fragment bands are present in the lane of particular restriction enzyme digest?
Explain.

Questions:
1. Name two methods that you used in lab to stop restriction enzyme digestions. Explain
mechanism of each.

2. a) State the principle of linear double stranded DNA separation by agarose gel
electrophoresis. Relate to visual appearance of 1 kb Plus ladder.
b) Present a completely labelled diagram of plasmid xyz digested with the restriction
enzyme Kpn1. There are two Kpn1 restriction sites in plasmid xyz. A complete Kpn1
digestion of plasmid xyz gives three bands; 3 kb, 0.5 kb, 1.2 kb. In the diagram include a
lane that represents (i) complete digestion of plasmid xyz and (ii) partial digestion of
plasmid xyz. Diagram must include accurate relative intensity of each band and be drawn
to scale (ie relative to 1 kb Plus ladder).

3. Summit a question about any part of this experiment that you do not understand. Questions
will be collected and discussed during Lab exam question period, week 11.
 38

LAB 4 DNA SEQUENCING OF RECOMBINANT PLASMID

Week 9

Part I: Sequencing Reaction of Recombinant Plasmid DNA

The following procedure will be preformed using pM13cam7Cla2.1 plasmid prepared in lab. Each
group will carry out one sequencing reaction using pM13cam7Cla2.1 DNA.

(a) Denaturation of the Double Stranded DNA

1. Add 5 :g pM13cam7Cla2.1 plasmid DNA to an eppendorf tube, adjust the final volume to
32 :l with sterile distilled water. Maximum 32 :l pM13cam7Cla2.1.

2. Add 8 :l freshly prepared 2 M NaOH. Mix by vortexing and quick spin. Incubate 10 min
at 37oC.

3. Premix 10 :l 3 M sodium acetate (pH 4.8 to 5.2) and 150 :l absolute ethanol. Quickly add
150 :l acetate-ethanol mixture to the denatured DNA and mix by vortexing.

4. Put at -20oC for 20 min. Spin* 15 min at 4oC. Aspirate off supernatant. Add 1 ml ice cold
absolute ethanol. Aspirate off ethanol. Wash once with 70% ethanol by carefully adding
200 :l 70% ethanol, do not disturb the pellet. Microfuge at 4oC for 1 min. Aspirate off
ethanol. Microfuge 10 second to bring down any remaining ethanol. Aspirate off ethanol.
To completely remove remaining ethanol aspirate until pellet is dry or heat tube at 65oC for
3 min with an open lid. NO ethanol should remain. Why?
*TIP. When centrifuging any liquid where you are collecting a pellet in an eppendorf tube
alway place the tube in the microfuge with lid joiner facing outwards. So when you
remove your tube from the microfuge you know where to look for the pellet; at the botton
of the tube below lid joiner. This allows you to keep the aspirator tip away from the pellet
area if the pellet is poorly visible.

5. Dissolve DNA in 10 :l sterile distilled water by vortexing for 1 min. Quick centrifuge to
bring liquid to bottom of tube.

(b) Annealing Template and Primer

6. For each set of four sequencing lanes, a single annealing reaction is used. Set up annealing
reaction by adding the following to tube containing 10 :l denatured DNA:
Primer (T7 primer for our reaction) 10 ng/:l 2 :l
Annealing buffer 2 :l

Short vortex to mix, followed by a quick spin in the microfuge.

7. Incubate in 65oC waterbath for 5 min. Quickly transfer to 37oC waterbath for 10 min. Let
sit at room temperature for 5 min. Proceed immediately to sequencing reaction.
 39

(c) Labelling Reaction

8. This step is done by the instructor ONLY. (T7 DNA Polymerase is very expensive.)
T7 DNA Polymerase dilution: Dilute stock T7 DNA polymerase 1:4 in ice cold enzyme
dilution buffer just before required. Stock T7 DNA polymerase should be stored at
-20oC at all times, only remove it to quickly to make dilution. Return stock T7 DNA
polymerase to freezer immediately.
Diluted enzyme must be stored on ice.

9. Add the following to the annealed template-primer tube:

"-35S-dATP 1 :l
Labelling Mix-dATP 3 :l
Diluted T7 DNA polymerase* 2 :l
Quick spin to mix. Incubate at room temperature for 3 min.
*
Remember to never hold tube by bottom, always the top, away from enzyme.

(d) Termination Reaction

10. Add 2.5 :l of each appropriate ddNTP Mix-Short* to the side of labelled tubes (A, C, G,
T). It is best to prepare these tube just prior to step 7. Prewarm your tubes containing
ddNTP Mix-Short in a 37oC waterbath for 1 min.
*must use Mix-short* (50-500 bp) as it is the standard mix not Mix-long (100-1000 bp)
which is used only for sequence extension. Mix-long contains less ddNTP therefore longer
extensions before termination with ddNTP.

11. Add 4.5 :l labelling mixture to each tube. Quick spin to mix. Incubate at 37oC for 5 min.

12. Add 5 :l Stop Solution to each Termination Tube. Quick spin in microfuge. Heat
samples at 80oC for minimum of 2 min and immediately load 3 :l on polyacrylamide
sequencing gel. If unable to load all samples immediately, keep at 80oC until ready to load.
It is important that the samples are not allowed to sit at room temperature for an extended
period of time which allows renaturation of the single stranded DNA.
Note: If samples are not to be sequenced immediately, store at -20oC until ready to use.
When ready to sequence, heat at 80oC for minimum of 2 min and load immediately on
sequencing gel. Label samples carefully with group number, DNA type, and nucleotide
(A, C, G, or T) only as demonstrators will most likely be loading your samples.

Part II: Gel electrophoresis of DNA sequencing reactions and autoradiograph

[A demonstration of sequencing gel setup, running of gel, and sample loading will be
performed in lab. Actual loading of your samples (students may do this), running of gel,
drying of gel and exposing gel to film will be performed by the demonstrators.]

(a) Preparation of gel plates (Refer to Bio-rad manual in reference file for details)
[This will be demonstrated in lab]
1. Clean plates thoroughly with non-abrasive detergent solution.

2. Just before use, rinse plates thoroughly with deionized water followed by ethanol wipe.

3. Coat bottom plate, not top glass plate with Sigma Cote siliconizing solution. Add Sigma
Cote to plate by scattering three to four 100 :l amounts over surface. IT IS ESSENTIAL
THAT YOU WEAR DISPOSABLE GLOVES. Using kleenex rub in circular motion to
coat the plate surface with the siliconizing solution. Polish with kleenex. Wipe plate again
with ethanol (surface next to gel). DO NOT TOUCH SURFACE OF PLATES ONCE
THEY ARE CLEANED. HOLD BY EDGES.

4. Assembly of plate will be demonstrated in lab.

 40

(b) Preparation of gel solution

1. A commonly used DNA gel preparation protocol:

30% acrylamide stock solution:

(acrylamide:bisacrylamide, 19:1 w/v)
28.5 g acrylamide plus 1.5 g Bis-Acrylamide to a total volume of 100 ml deionized H2O.

Sequencing polyacrylamide gel system is 8% strength prepared with the following

components (75 ml required for narrow 6 sample gel):
30% (w/v) acrylamide 40 ml
(acrylamide:bisacrylamide, 19:1)
urea (ultrapure) 63 g
10x Tris-borate-EDTA, pH 8.3 15 ml
distilled water to 150 ml

2. Dissolve urea by incubating in 37oC bath and periodically shaking. Best to filter the
solution through a 0.45 micron mesh filter. Degas solution for about 15 min.

3. Casting tray Sealing Gel Protocols: for 21 cm gels mix 20 ml 8% acrylamide gel solution,
250 :l 10% ammonium persulfate, and 100 :l TEMED. Mix immediately, pour into tray.
Immediately place assembled glass plate sandwich in tray in a vertical position. Gel
polymerizes in 30 seconds, you have very little time to work. Let sit for minimum of 2 min
before adding gel solution. For gel solution: 40 ml acrylamide solution, 40 :l TEMED,
and 250 :l ammonium persulfate.

(c) Pouring of acrylamide gel

CAUTION: Liquid acrylamide is extremely dangerous, always handle with gloved hands.
After polymerization of the acrylamide caution need not be as stringent.

1. To prevent gel overflowing into the buffer chamber, fill 1/3 with 1x TBE buffer (dilute out
overflow, so will not polymerize) or stuff with paper towels.
2. Using a 50 ml syringe chamber, add gel solution between plates. Hold the plate sandwich
at a 30o angle so that the solution flows down along the side of one spacer. Be careful to
maintain an even flow of the gel solution to negate any bubble formation. If air bubbles
occur, place plates vertically and firmly tap to work bubble(s) up, return to 30o angle, and
continue filling plates. Fill plates until the liquid reaches the top edge.
3. Carefully insert the sharks-tooth comb(s) flat edge first into gel solution to a depth of 2-3
mm below the short plate.
4. Leave the plates in this position for 30-60 min so that the gel polymerises. For best
resolution pour the plates at least 3 hours before running. The plates may be poured the
day before using. Leave at room temperature overnight, but to keep the gel from drying at
the top, carefully fold 1/2 thick layer of wetted paper towels over the top. Cover towels
with saran wrap and tape into place with masking tape to prevent evaporation.
5. To use gel, wash off acrylamide and urea from the surface of the plates and in the comb
area.
6. Carefully slide comb out (DO NOT MOVE FROM SIDE TO SIDE). Rinse the top of the
gel with deionized water, rinse comb and re-insert with the teeth down toward the gel.
Insert comb until it just makes contact with the gel surface without piercing the gel. A
slight indentation of the gel surface is evident if the comb has been properly inserted. DO
NOT SLIDE COMB LATERALLY IN ANY OF THESE MANIPULATIONS.

C. PRE-ELECTROPHORESIS/ELECTROPHORESIS OF SEQUENCING GEL

CAUTION: Although equipped with safety lids/covers, use electrophoresis system with
extreme caution. CARELESS OPERATION CAN RESULT IN ELECTROCUTION.
 41

Never: operate damaged or leaky equipment

Always: turn off power supply before disengaging power cord

(a) Pre-electrophoresis

1. Secure gel plates in position in the apparatus for sequencing taking care to ascertain that
vulnerable positions are free of liquid leaks.

2. Fill lower buffer chamber with 350-500 ml 1x TBE buffer. Do not fill the lower chamber
with more than 500 ml. Fill the upper buffer chamber with buffer using the flared portion
of the panel as a fill spout. The level of the buffer should be about 0.6 cm from the top of
the fill spout at all times. The upper buffer must cover the top of the acrylamide gel.

3. Cover with safety lid(s) and connect to power supply. Electrophoresis at 35 mamp for 30
to 60 min before loading DNA sequencing samples. The temperature should be 50oC
before loading samples.

(b) Sample application and electrophoresis

1. Prepare freshly heated and chilled DNA sequencing samples as described earlier.

2. After pre-electrophoresis, load gel immediately with DNA sequencing samples as follows.
It is very important that you rinse out wells of the gel formed by the sharks-tooth comb
using a Pasteur pipette just before loading samples. This displaces urea solution from the
gel surface giving flat compact bands.

3. Using a P20 pipetman, apply 3 :l of sequencing reaction product to the arch of the comb
between the two plates (under buffer). APPLY CAREFULLY. DO NOT SQUIRT. Put
the A, C, G, and T reaction products in adjacent wells. WORK QUICKLY.

4. Close equipment and restart electrophoresis with the same voltage as for pre-
electrophoresis. For Bio-Rad sequencing gel (21 x 50 x 0.4 cm), set at constant mamp of
30-33 using 2000 volts.

5. Follow the course of electrophoresis via the tracking dyes, xylene cyanol and bromophenol
blue. When the bromophenol blue dye reaches the bottom of the gel, the electrophoresis
can be stopped. The DNA base read at the position is approximately 20 bases from the 5'
end of the DNA chain synthesized. Xylene cyanol which would have migrated about
three-fifths of the length of the gel will signify where the 70th base is from the 5' end.
Depending on what you already know about the DNA sequence, you can electrophoresis
further until xylene cyanol is at the bottom of the gel and this will
increase the length of sequence you can decipher in the upper section of the gel.
 42

(c) Disassembling the sequencing gel

1. Turn off power supply and disconnect apparatus. WEAR GLOVES.

2. Pour off buffer from upper chamber (small unit) or open drain for larger electrophoresis
unit.

3. Carefully remove plates and place on bench area covered with protective paper making
sure that none of the buffer contaminates the floor or bench - it is radioactive.

4. Using a thin spatula, carefully prise sialanised plate (bottom) from the other plate (top) to
which the gel should stick. BE VERY CAREFUL IN DOING THIS OR THE GEL WILL
TEAR. Gel must stick to top plate as 35S-labelled gel must be fixed before exposing to
film. If gel is not sticking to top plate, try another corner. Also using a squirt water bottle
helps remove gel from plate while slowly lifting other plate.

5. Leaving gel on the top plate, place in large shallow tray with gel side up. Carefully pour
fixative solution (10% acetic acid: 10% ethanol) into tray (off to the side of the gel) and
allow it to rise and cover the gel. Let stand for at least 15 min with periodic gentle
agitation by hand. During this period, carefully work fixative so that it lifts the gel from
the glass plate but does not float away.

6. Shielding the gel slab with one hand, aspirate the majority of the fixer. Apply a precut 3
MM Whatman filter sheet on top of the gel and gently pat it so that it is completely wet.
Cover with several layers of paper towel and blot as much moisture as possible, especially
around the edges. Discard paper towels. After ensuring that the gel has adhered to the
Whatman sheet, carefully peel the gel attached to paper back from the glass plate and put in
pre-heated vacuum gel dryer (gel upwards), cover with plastic sheet, and dry for 20-30 min
at 80oC.

D. AUTORADIOGRAPHY

The dried gel is placed in a X-ray film cassette and exposed to a suitable X-ray film overnight at
room temperature. Develop film.

Week 10

Analysis of sequencing gel results and lab exam outline.

Week 11
Exam Question Discussion Lab

1. Summit a question about any part of this experiment that you do not understand. For
example, why add urea to the acrylamide. Questions will be collected at the start of lab.
 43

LAB REPORT

Data Analysis
1. Include a completely labelled figure of the autoradiogram (or photocopy or scan printout)
of sequencing gel.

2. Include a photocopy of Figure 4 from reference LéJohn, H.B. 1989. Structure and
Expression of Fungal Calmodulin Gene. Journal of Biological Chemistry.
264:19366-19372. Highlight your sequence. Note any differences, but remember when
your are reading your sequence from the x-ray film scanned printout, read only as far as
can accurately read.

3. Present a completely labelled figure of BLAST alignment search results of your sequence.
Present only the highest scoring alignment. Search website: www.ncbi.nlm.nih.gov
Select BLAST search and follow instructions on website. To save paper cut and paste this
alignment to a wordprocessor program. Block and convert to Courier 10 font (all symbols
are equally spaced, so it will appear the same as the website). What is the accession
number?

Questions

1. In the fungal calmodulin paper by LeJohna, 1989, the complete gene including upstream
and downstream sequences was sequenced using two plasmid clones, pM13cam7Cla2.1
and pM13cam7Cla1.7. In your lab you were able to accurately sequence approximately
200 bp of the coding sequence.
a) What was the sequencing strategy used by LeJohn to completely sequence both
plasmids, pM13cam7Cla2.1 and pM13cam7Cla1.7 and correctly align these clones?
b) Briefly outline another possible sequencing strategy. Do not include experimental
details.
Note: in research paper by LeJohn the Cla2.1 fragment is noted as 2.0 kb.
a
LéJohn, H.B. 1989. Structure and Expression of Fungal Calmodulin Gene. Journal of Biological
Chemistry. 264:19366-19372.

2. a) The sequencing gel is pre-run for at least 30 minutes before loading DNA samples.
Explain why.
b) When placing the comb on gel for DNA sample loading, it is important not to press
comb teeth into the gel. Explain why.

3. Explain why acrylamide, not agarose must be used to sequence DNA.

4. What is the function of each component of DNA polymerase enzyme dilution buffer: 20
mM Tris-HCl, pH 7.5, 5 mM DTT (dithiothreitol), 100 :g/ml BSA (bovine serum
albumin), and 5% glycerol.
 44

APPENDIX
SAMPLE CALCULATION of TITER - sample given below is for bacteria/ml. However, the
method of calculation is identical for phage/ml.

SAMPLE CALCULATION of the number of bacteria per ml

Data for example calculations using the following sample data
Dilution plated Number of colonies
Plate 1 Plate 2
10-2 TNTC TNTC
10-3 320 316
10-4 34 27
10-5 2 3
TNTC = too numerous to count

Terms
Plating factor = reciprocal of volume plated
Dilution factor = reciprocal of dilution for significant counts
Significant plate counts = the sum of the plate counts at significant dilution divided by number of
plates. Often more than one dilution has significant plate counts. It is important to use all
significant plate count data. There are several ways to deal with data that has more than one
significant plate count dilution.
Number of plates = number of significant plates

Calculation
Do not average an average value as it incorporates error in your calculation (not statistically
accurate). Use one of the following methods to calculate bacteria titer.

Bring all significant counts to the same dilution:

Bacteria/ ml = significant plate counts x dilution factor x plating factor

number of plates

(320 + 316 + 340)/3 x 1/10-3 x 1/10-1 = 3.25 x 106 bacteria/ml, since the smallest number of
significant figures for plate counts is two, the answer is 3.3 x 106 bacteria/ml

Or calculate the titer for each significant plate count and average.

Bacteria/ml = significant plate count x dilution factor x plating factor

320 x 1/10-3 x 1/10-1 = 3.20 x 106 bacteria/ml

316 x 1/10-3 x 1/10-1 = 3.16 x 106 bacteria/ml
34 x 1/10-4 x 1/10-1 = 3.4 x 106 bacteria/ml

Average all values: (3.20 x 106 + 3.16 x 106 + 3.4 x 106)/3 = 3.25 x 106 bacteria/ml, since the
smallest number of significant figures for plate counts is two, the answer is 3.3 x 106 bacteria/ml
 45

MEDIA

LB medium: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 800 ml distilled water.
Adjust to pH 7.5. Bring volume to 1 litre. For plates add 15 g agar per litre.

LB-AMP medium: Add 100 :g/ml ampicillin.

SOLUTIONS

General

BRL restriction enzyme buffers, example:

REact 1: (1x) 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2

TE buffer, pH 7.5: 10 mM Tris-HCl, pH 7.5, 1 mM Na2EDTA

TE buffer, pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM Na2EDTA.

Tris-borate, pH 8.3: prepared as a 10x stock solution (stored 4C) as follows: Add 109 g Trisma,
55 g Boric acid, and 40 ml 0.5 M Na2EDTA (pH 8) per litre distilled water. The pH should be
approximately 8.3.

TAE buffer (10x): 48.4 g Trisma base, 11.4 ml glacial acetic acid, 20 ml 0.5 M EDTA, pH 8.0,
and distilled water to 1 liter.

Saline: 8.5 g NaCl and distilled water to 1 liter.

Agarose stop solution: Dissolve 12 g urea, 25 g sucrose, 0.95 g Na4EDTA, and 0.5 g bromophenol
blue in 40 ml distilled water. Adjust to pH 7.0. Bring volume up to 50 ml with distilled water.

Phage buffer (SM BUFFER ):5.8 g NaCl, 2.0 g MgSO4.7H2O, 50 ml Tris HCl (1 M stock sol. pH
7.5), 0.5 ml Gelatin (2% stock sol.). Make up to 1 liter with distilled water.

STE buffer: 100 mM NaCl, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA

TFB buffer: 10 mM K-MES (pH 6.2), 45 mM MnCl2.4H2O, 10 mM CaCl2.2H2O, and 3 mM

Hexamine cobalt chloride. Stock solution,1 M MES is adjusted to pH 6.3 with KOH, filter
sterilized, and store at -20oC.

T4 Ligase reaction buffer: 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 1 mM ATP, 1 mM DTT, 5%

(w/v) polyethylene glycol-8000 (PEG).

T4 ligase enzyme storage buffer: 10 M Tris-HCl (pH 7.5), 5 mM KCl, 1 mM DTT, 50% (v/v)
glycerol.

Plaque Blotting:
Denaturing solution: 1.5 M NaCl, 0.5 M NaOH
Neutralizing solution: 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2, 1 mM Na2EDTA

20x SSC: 175.3 g NaCl + 88.2 g sodium citrate per liter distilled water. Adjust the pH to 7.0
with few drops of 10 N NaOH. Adjust to final volume of one liter. Sterilize water.. Dilute with
distilled water for required dilution.
 46

Plaque Blot Hybridization

Hybridization Solution: 0.5% [w/v] SDS sodium dodecyl sulfate (also called sodium lauryl
sulfate), 5x Denhardts, 6x SSC

100x Denhardts Solution: 2.0% Ficoll, 2.0% polyvinylpyrrolidone (PVP), 2.0% BSA (bovine
serum albumin)

Promega mini-column DNA preps:

Cell Resuspension Solution: 50 mM Tris-HCl, pH 7.5, 10 mM EDTA,
100 :g/ml RNase A
TE Buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
Cell Lysis Solution: 0.2 M NaOH, 1% SDS
Column Wash Solution: 200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA...dilute
1:1 with 99.9% EtOH
Neutralizing Solution: 2.55 M KOAc, pH 4.8

Prep-A-Gene-DNA Purification System:

Binding Buffer: 6 M sodium perchlorate; 50 mM Tris-HCl (pH 8.0); 10 mM EDTA (pH
8.0)
Wash Buffer + EtOH: 400 mM NaCl; 20 mM Tris-HCl (pH 7.5); 2 mM EDTA (pH 7.5);
50% EtOH (v/v)

CONCERT rapid plasmid purification system:

Cell Suspension Buffer (G1): 150 mM Tris-HCl, pH 8.0, 10 mM EDTA,
100 :g/ml RNase A
TE Buffer (TE): 10 mM Tris-HCl, pH 8.0, 1 mM EDTA
Cell Lysis Solution (G2): 0.2 M NaOH, 1% SDS
Wash Buffer (G4): 200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA...dilute 1:1 with
95% EtOH
Optional Wash Buffer (GX): product proprietary formulation (contains acetate, guanidine
hydrochloride, EDTA and ethanol)
Neutralizing Buffer (G3) product proprietary formulation (contains acetate and guanidine
hydrochloride)

Sequencing solutions:
DNA polymerase enzyme dilution buffer: 20 mM Tris-HCl, pH 7.5, 5 mM DTT
(dithiothreitol), 100 :g/ml BSA (bovine serum albumin), and 5% glycerol.
Annealing buffer: 1 M Tris-HCl, pH 7.6, 100 mM MgCl2, and 160 mM DTT.
Labelling Mix-dATP: 1.375 :M each dCTP, dGTP and dTTP and 333.5 mM NaCl.
`A' Mix-short: 840 :M each dGTP, dCTP, and dTTP; 93.5 :M dATP; 14 :M ddATP; 40
mM Tris-HCl (pH 7.6) and 50 mM NaCl.
`G' Mix-short: 840 :M each dATP, dCTP, and dTTP; 93.5 :M dGTP; 17 :M ddGTP, 40
mM Tris-HCl (pH 7.6) and 50 mM NaCl.
`T' Mix-short: 840 :M each dGTP, dATP and dCTP; 93.5 :M dTTP; 14 :M ddTTP; 40
mM Tris-HCl (pH 7.6) and 50 mM NaCl.
`C' Mix-short: 840 :M each dGTP, dATP and dTTP; 93.5 :M dCTP; 17 :M ddCTP, 40
mM Tris-HCl (pH 7.6) and 50 mM NaCl.
Stop solution: 10 mM EDTA, pH 7.5, 97.5% formamide, 0.3% bromophenol blue and
0.3% xylene cyanol FF
 47

EQUIPMENT OPERATION

PIPETMAN OPERATION

In your lab, you have available three different pipetmen depending on the lab. If you look at the
top of the plunger it states the size of the pipetman. P20 measures accurately from 2 :l to 20 :l.
P200 measures accurately from 20 :l to 200 :l. P1000 measures accurately from 100 :l to 1000
:l. Never turn the pipetman above the maximum volume; 20 :l for P20, 200 :l for P200, and
1000 :l for P1000 as this breaks the pipetman. The scale on the pipettor is read different for each
type - refer to Figure 5 for an example of how to read the scale.

(Excerpted from Gilson pipetman operation manual.)

1. Setting the volume: The required volume is set on the digital volumeter by turning the
knurled adjustment ring (Figure 7-2A). When the volumetric setting is increased, it is
necessary to go about 1/3 of a turn above the desired setting and then come back to the
exact value. When the volumetric setting is decreased the desired value may be selected
directly. The volumeter display is read from top to bottom in :l for P20 and P200 and ml
for P1000 (Figure 7-2).

2. Place a disposable tip on the shaft of the Pipetman. Press on firmly with a slight twisting
motion to ensure an airtight seal. Depress the push-button to the first positive stop (Fig. 7-
3A). While holding the Pipetman vertical, immerse the tip 2-4 mm into the sample liquid.
Release the push-button slowly to draw up the sample (Fig. 7-3B). Wait 1 to 2 seconds,
then withdraw the tip from the sample.

3. To dispense the sample, place the tip end at a 10-45o angle against the inside wall of the
vessel and depress the push-button SMOOTHLY to the first stop (Fig 7-3C). Wait 1 to 2
seconds and then depress the push-button completely to expel any residual liquid (Fig. 7-
3D). With the push-button fully depressed, carefully withdraw the Pipetman, sliding the
tip along the inside wall of the tube. Release the push-button. Remove the used tip by
depressing the tip ejector button (Figure 7-1F).
 48

Figure 7: Gilson pipetman operation.

1-A, push-button; 1-B, moulded hand grip; 1-C, shaft; 1-D, built-in ejector; 1-E, tip; 1-F,
ejector button; 2-A, knurled adjustment ring; 3-A, 3-B, 3-C, and 3-D as discussed in
operation of push-button.
 49

BIO-RAD DNA SUB-CELL agarose gel electrophoresis system

Running buffer: Use approximately 1.5 - 2 liters 1x (TAE) Tris-acetate buffer, such that the
surface of gel is covered by a minimum of 3 mm of buffer. Students are supplied with 10x stock
of TAE buffer. Dilute to 1x before using.

Ethidium bromide staining: the gel may be stained bp

during electrophoresis, by adding 0.5 :g/ml EtBr (10 1 2 ,2 1 6
1 1 ,1 9 6
mg/ml stock) to the running buffer or stained after 1 0 ,1 8 0
running by placing gel in 1x Tris-acetate buffer 9 ,1 6 2
8 ,1 4 4
containing 0.5 :g/ml EtBr for 15 min, then rinse with 7 ,1 2 6

water. Or best of all, add 7 :l 10 mg/ml ethidium 6 ,1 0 8

5 ,0 9 0
bromide to 200 ml agarose gel solution just before 4 ,0 7 2

pouring. 3 ,0 5 4

Agarose gel: Prepare required percentage agarose in 200 2 ,0 3 6

1 ,6 3 6
- 300 ml* 1x Tris-acetate buffer. Heat mixture while
stirring on a heater until comes to a boil and is completely 1 ,0 1 8
dissolved. Cool to 55oC and pour into agarose holder that
has been taped at ends with masking tape and well maker 506, 517
positioned. Allow to set at room temperature for 20 min. 396
344
* The volume depends on size of agarose gel bed and 298
whether preparative (usually greater depth) or analysis 220
201
gel. 154
134
75
Set up of agarose gel electrophoresis unit: Remove
tape from ends of agarose gel holder and place gel in F ig u re 8 . 1 K b D N A L a d d e r F ra g m e n ts . F ra g m e n ts a re

electrophoretic unit containing running buffer. Remove lin e a r d o u b le s tra n d e d D N A , th e re fo re c a n o n ly u s e a s a

s ta n d a rd fo r lin e a r d o u b le s tra n d e d D N A . B a n d s a re
the comb before putting gel in electrophoretic unit. v is u a liz e d b y e h tid iu m b ro m id e s ta in in g .

DNA standard: The 1 Kb ladder or 1 Kb ladder Plus are

frequently used as linear double stranded DNA standards
bp
12,000 (figures 8 and 8a from Gibco BRL Catalogue). The standard
11,000 can only be used to determine the size of linear double
10,000
9,000 stranded DNA fragments.
8,000
7,000
6,000 Sample Preparation and Loading: DNA samples
5,000 containing 'stop' (16.7%) solution should be placed at 65oC
4,000
3,000 for 5 min then cooled to room temperature before loading
into agarose gel wells if stored at -20oC. The 1 kb Plus
2,000 ladders needs only to be thawed at 37oC before loading on
1,600 the gel. Add 3 :l 'stop' solution per 15 :l sample. Each
sample is loaded using an eppendorf micropipet.
1,000
850
650 Running of agarose gel electrophoresis: The power supply
should be connected such that the negatively charged DNA
500
400 will migrate to the positive electrode.
300 Running times: 80-100 volts for 3 hours at room
200
100 temperature, 15-25 volts overnight at room temperature
(depends on temperature and volume of running buffer).

Figure 8a. 1 Kb Plus DNA Ladder Fragments. Fragments are

linear double stranded DNA, therefore can only use as a
standard for linear double stranded DNA. Bands are
visualized by ehtidium bromide staining.
 50

OPERATION OF FLOOR MODEL CENTRIFUGES

Note: If procedure varies depending on centrifuge manufacturer a step by step operation
procedure is usually located on or nearby the centrifuge or the teaching assistant will help you.

HITACHI HIGH SPEED HIMAC REFRIGERATED CENTRIFUGE

• to select or change settings the CHECK button must first be pressed (light on). The light
stays on for 16 sec. When the light is off you can no longer select, change setting or carry
out any operation, just press check button again and continue.
• When the centrifuge is turned on and the CHECK button is not pressed. The centrifuge
displays real time parameters.

OPERATION
Centrifuge tubes should be balanced by scale by adding or removing appropriate solution from one
of the tubes.

1. Turn power switch on. The indicators on the control panel are illuminated. The door lock
is released.
2. Open door. If required set the rotor gently in position and close door. Turn the rotor lightly
by hand to check that the rotor is correctly set. Remove the rotor lid and place balanced
tubes opposite each other in rotor. You cannot run the centrifuge with an odd number of
tubes. SCREW ON LID.
3. Call up memory code number or enter parameters.
Call up pre-programmed memory code number: Press CHECK button, MEMORY button,
memory code number, and CALL button. Each memory code number consists of a
specified set of operation parameter (see sheet on centrifuge cover). See below for a list of
operation parameters and how to set and store operation parameters.
OR
Real time operation (enter original parameters): see setting of operation parameters below.
4. After the parameters are set make sure the check light is still on. If not, press the CHECK
button.
5. Press the START button. The rotor starts running. The start lamp begins flashing. The
timer starts to count down.
6. The timer counts down to zero or press the STOP button. The rotor begins to decelerate.
The stop light begins flashing.
7. The rotor stops. The stop light stops flashing. A buzzer sound occurs. The door lock is
released.
8. Unscrew rotor lid and remove tubes. If required, use tweezers to help remove tubes. Wipe
out rotor if spills occur. DO NOT SCREW ON THE LID just place on top of the rotor.
9. Close centrifuge lid and turn off power.

PARAMETERS
ROTORS NUMBER: SMALL (maximum volume 40 ml) RPR20-2 = ROTOR #7
LARGE (maximum volume 450 - 500 ml) RPR9-2 = ROTOR #13
TEMPERATURE: 4 to 20 oC
SPEED: Rotor number 7 (small) - maximum speed 18,000 rpm
Rotor number 13 (large) - maximum speed 8,000 rpm
Example: for 3,520 rpm, press 3 . 5 2
TIME 0 to 99 min 59 sec, FREE
Example: for 5 min and 30 sec, press 5 . 3 0
ACCEL. Higher the value the faster the acceleration - 9 is good for basic
centrifugation.
DECEL. Higher the value the faster the deceleration - 7 is good for basic
centrifugation.
Example: for loose pellet or phase separation the deceleration number
should be decreased to 3.
 51

SETTING OF OPERATION PARAMETERS

1. Press CHECK button, CHECK button lights up. Parameter for preceding operation are
displayed. The LED light goes off after 16 sec, it is only possible to set parameters with
the CHECK button light on. Press CHECK again if light goes out.
2. Press TEMP button. Position where setting is to be made will flash. Using the ten number
key pad, key in desired value for parameter setting. Data will be displayed in the above
setting position. Press SET button. The flashing stops and selected operation parameter is
set.
Note: if your require 4oC, set at 8oC as the centrifuge often goes below the requested
setting. Your sample may freeze if spinning for greater than 5 min at a high speed.
3. Repeat step 2 for each remaining parameter...SPEED, TIME, ROTOR NO., ACCEL., &
DECEL. Remember to press the CHECK button again if the CHECK light goes out.

STORING OF OPERATION PARAMETERS (Storing of operation parameters is optional.)

4. After setting desired parameters as above, press CHECK if light has gone out.
5. Press MEMORY, memory code number (one that is available) and the RECORD button.
There are nine available memory program code numbers. See chart on centrifuge lid for
memory programs that already exist.
 52

REFERENCES

Any good molecular biology text or lab manual as not all background information available in the
reference file.

References available in lab Reference Binder in the Science and Technology Library (1 hour
reserve).

# ARTICLE/SOURCE
_________________________________________________________________
1 Ausubel, F., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., & K. Struhl, 1992. Lambda
as a cloning vector and Lambda phage and DNA Preparation . In: Short Protocols in Molecular Biology,
2nd edition. John Wiley and Sons. p 1-30, 1-31 and 1-39 to 1-45.

2 Berger, S.L. & A. R. Kimmel. 1987. Bacteriophage 8 and preparation of genomic library in 8 . Methods
in Enzymology: Guide to Molecular Cloning. 152:183-199.

2a Karcher, SJ 1995. Southern blot analysis. in Molecular Biology: A project approach. Toronto: ASM p.135-
161

3 [Anonymous] Protocols for nucleic acid blotting and Hybridization. Amersham Instruction Manual -
Hybond-N+. p 1-10.

3a Sambrook, J., Fritsch, E.F. & T. Maniatis. 1989. Agarose Gel Electrophoresis. In Molecular Cloning: A
Laboratory Manual, 2nd ed. Plainview: Cold Spring Habor Laboratory Press. p 6.2-6.7, 6.13, 6.15.

4 [Anonymous] 1986. Comparison of Electrophoretic Migration of Linear and Supercoiled Molecules.

Focus 8.3: 3-4.

5 [Anonymous] 1998. Concert rapid plasmid purification system. Life Technologies, Gibco BRL.

6 [Anonymous] Prep-A-Gene® DNA Purification Systems. Instruction Manual. Bio-rad. p. 1-3.

7 [Anonymous] 1987 Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate. Focus
9.2: 3-5.
8 [Anonymous] 1986 Ligation Update: Insert to Vector Ratio. Focus 8.4:12.

9 [Anonymous] 1986. Optimizing DNA Ligations for Transformations. Focus 8.1:1-3.

10 [Anonymous] 1986-87. Cloning Strategies. Life on the Edge. 2:86-88.

11 Hanahan, D. 1996 Mechanisms of DNA Transformation. In: Neidhardt FC, editor. Echerichia coli and
Salmonella typhimurium, 2nd ed., Washington: ASM Press. p 2449-2459.

11a Karcher, SJ 1995. Molecular Biology: A project approach. Toronto: ASM p. 63-88

12 LéJohn, H.B. 1989. Structure and Expression of Fungal Calmodulin Gene. Journal of Biological
Chemistry. 264:19366-19372.

13 [Anonymous] T7 Sequencing Kit™. Instruction Manual. Pharmacia Biotech. p. 1-36.

14 [Anonymous] 1993 Sequi-Gen® Nucleic Acid Sequencing Cell. Bio-Rad Instruction Manual. p 5-19.

15 Sambrook, J., Fritsch, E.F. & T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed.
Plainview: Cold Spring Habor Laboratory Press.p 13.3 - 13.20
 53

Recombinant DNA Technology 60.457 FINAL LAB EXAM

DATE: sample PAGE: 1 of 2 TIME: 1.5 h
INSTRUCTOR Dr. L. Cameron
Student Name __________________________ Student Number ______________________
EXAMPLE LAB EXAM FOR REFERENCE BINDER.
Answer exam questions in PEN ONLY.
Answer QUESTIONS ON EXAM PAPER. Spacing has been removed for example exam.

10 1. Briefly answer each of the following questions.

a) What is the purpose of the E. coli tranformation experiment in your recombinant DNA
lab with reference to Achlya calmodulin gene?
b) Two DNA samples, restriction digested with Cla1, were run on agarose gel
electrophoresis. Explain why the intensity of Cla1 2.1 fragment differs for each DNA
sample.
Experimental information:
Achlya DNA samples: (1) pM13cam7Cla2.1 DNA and (2) EMBL3 lambda clone cam7
DNA
10 :l of each DNA loaded.
The concentration of each DNA sample is 0.2 :g/:l.
c) What reaction does DNA polymerase I/DNase I catalyze?
d) Plaque blots are placed in denaturing solution during the preparation of genomic library
plaque blots. Explain the function of the denaturing solution.
e) The annealing reaction for plasmid DNA sequences is incubated in a 65oC waterbath for
5 min. Quickly transferred to a 37oC waterbath for 10 min. Let sit at room temperature for
5 min. These steps are performed immediately before the sequencing reaction. Explain
what is happening at the molecular level.
f) Acrylamide sequencing gel results are of single stranded DNA fragments. What
procedures are performed and/or components added to produce and maintain single
stranded DNA fragments?
g) When reading results from a sequencing gel loaded left to right A, C, G, T the gel is
usually read bottom up. Explain why and how to read the sequence correctly. Explanation
must include schematic diagram of sequencing gel film.
h) Agarose gel electrophoresis is repeatedly used in the recombinant DNA lab. What is the
principle of agarose gel electrophoresis for separation of linear double stranded DNA?
i) What procedure(s) make it possible to isolate ONLY plasmid DNA from E. coli cells?
j) What is stringent plaque blot washing conditions. Explain with reference to principles
involved.

7 2. Explain the function of the following solutions, media, or components that were used in the
recombinant DNA lab.

a) ddCTP in sequencing reaction

b) LB broth
c) denhardts solution
d) 300 mM Na2EDTA, pH 8.0; nick translated
e) plasmid DNA spin cartridge
f) TE buffer, pH 8.0
g) ethanol in DNA purification

CONTINUED ON PAGE 2...

 54

Recombinant DNA Technology 60.457 FINAL LAB EXAM

DATE: sample PAGE: 2 of 2 TIME: 1.5 h
INSTRUCTOR Dr. L. Cameron

Student Name __________________________ Student Number ______________________

8 3. Briefly answer each of the following questions.

a) State features of pM13 that make this plasmid a good cloning plasmid for eukaryotic
genomic DNA.

b) Plasmid DNA is prepared by rapid phenol extraction method in your lab. State the
purpose of this experiment. Present a schematic labelled diagram of agarose gel
photograph showing the desired results.

c) Outline an experiment to select DNA fragment to clone into pM13. A positive genomic
lambda library clone has already been isolated. Do not include procedure to isolate DNA
fragment.

d) The order of restriction sites in the multi-cloning site of pM13 (3 kb) is T7 primer-
Not1, Xba1, Spe1, BamH1, Sma1, Pst1, EcoR1, EcoRV, Hind111, Cla1, Sal1, Acc1,
Xho1-T3 primer. The genomic DNA is inserted into the Pst1 site.
Restriction enzyme digestion produced the following fragment kb sizes.
Restriction enzyme Fragment size (kb)
Acc1 4.0, 0.8
BamH1 4.5, 0.3
EcoR1 3.6, 1.2
Not1 4.1, 0.7
Pst1 3.0, 1.8
Present a restriction map of the genomic DNA with correct orientation in pM13 plasmid.

3 4. Determine the ratio of DNA molecules that are successful at transformation. The following
transformation data and results were obtained for a recombinant pM13 transformation
experiment.
DATA: Transformation mix - 10 :l ligation mixture (pM13-genomic insertl), 50 :l
transformation buffer, 100 :l competent cells, then 0.4 ml LB broth added before plating
0.1 ml of the following dilutions of this mixture.
Dilution LB-AMP-X-gal-IPTG
Total colony count
plate 1 plate 2
_________________________________
10-1 342 318
10-2 32 34
10-3 2 1
10-4 0 0
TNTC = too numerous to count
LB-AMP = LB agar containing ampicillin
Molecular weight of recombinant pM13 =2.8 x 106.
The concentration of pM13-genomic insert is 0.024 :g/:l.

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