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BioC 4025W Fall 2017 Lab 7

Lab Manual LAB 7

PROTEIN CONCENTRATION
CHECKING RESULTS OF LIGATION/TRANSFORMATION
INQUIRY: What is the probability that your colonies would have the insert if you don’t
have blue/white screening to help you identify them? How could you estimate this?

Protein determination – Refer back to Lab Manual 3 for a description of protein many
different protein determinations that have been used through the history of biochemistry.
http://wolfson.huji.ac.il/purification/Protocols/Comparision_of_Methods.htm

The importance of the protein determination in our purification of the LDH enzymes
(beef heart and barracuda) is that it enables us to calculate the specific activity of the
LDH. The specific activity (SA – product formed / unit time / mg protein) is our ultimate
measure of purification of active enzyme. To make your purification table, you need to
determine the amount of protein in samples you took at each step of the purification,
and then back calculate the total protein in each step. This is the total (bulk) protein, of
which, as you go through the purification will contain a larger and larger fraction of LDH.

Ligation – The ligase (usually from


T4 even phage) reaction joins the
vector DNA ends with those of the
insert. The reaction is facilitated by
the generation of overhangs that can
associate due to base pair
complementarity. The reaction
requires ATP. What are the
overhang sequences produced by
restriction by NdeI and BamH1?

Transformation – Transformation is the process of taking up DNA by bacteria (E. coli).


Bacteria are made leaky by incubating them with CaPO4 at low temperature (where the
membranes are not fluid), then adding the ligated plasmid, and then bringing the
temperature up to allow the membranes to seal. This traps the plasmid DNA inside the
bacteria. This is a rare event, so a selection system is needed to know whether the
plasmid has been taken up. For this, antibiotic resistance is used. On the plasmid
there is a gene that can make a protein that degrades the antibiotic to allow the bacteria
to grow. Beta-lactamase can degrade the lactam ring of ampicillin to confer resistance
(structures shown below).

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BioC 4025W Fall 2017 Lab 7

The blue white selection (see week 6 lecture and prelab question) also allows you to
know if your insert was ligated into the vector.

THE NEXT TWO PAGES CONTAIN Prelab questions for Lab 7

How can you select for cells that have the plasmid?
How can you screen for the plasmid containing the insert?

1. This question illustrates what you Additions Rxn 1 Rxn 2 Rxn 3


see on your plates after ligation and
transformation. It is, however, set up
with blue/white testing (see lecture 6), Plasmid Vector 5 µl 5 µl 2.5 µl
whereas your LDH transformations do (7170 bp)
20 ng/microliter
not have blue/white testing.
DNA Insert 0 2 µl 5 µl
You will assess the results of 3 ligation (1434 bp)
reactions shown in the table. ng/microliter
Reaction 1 - 20 femto (f) moles of 0.2 M TrisHCl 5 µl 5 µl 5 µl
vector alone; pH 8.0
Reaction 2 - 20 fmoles of vector and a 0.4 M MgATP 1 µl 1 µl 1 µl
2 fold molar excess of insert;
T4 ligase 5 µl 5 µl 5 µl
Reaction 3 - 10 fmoles of vector and a
200 U/microliter
10 fold molar excess of insert.
Water 84 µl 82 µl 81.5 µl
Vector = pBioC4025 digested with
Nde I and HinD III, size 7.17 Kb,
Insert = Nde I – HinD III digested DNA Total 100 µl 100 µl 100 µl
fragment, size 1.434 Kb. Volume
MW of a base pair = 700 Daltons

The plasmid vector you used had Blue/White (B/W) test capability and confirms Ampicillin
(Amp)-resistant. The E. coli cells are also engineered to be B/W capable. Set up and plate 6
transformations (3 ligation rxns and 3 controls).

For Lig Rxns 1-3, refer back to the ligation table

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BioC 4025W Fall 2017 Lab 7

Experimental Pre-lab questions continued


Transformed Lig Rxn 1 Transformed Lig Rxn 2 Transformed Lig rxn 3
IPTG + X-gal +Amp IPTG + X-gal +Amp IPTG + X-gal +Amp

Controls
Transformed with Competent cells Competent cells
Circular B/W plasmid +Amp no Amp
IPTG + X-gal +Amp

Assuming that your experiment worked, describe or draw what you see on each plate. If you
draw, use open circles [] for white colonies and filled circle [] for blue colonies. Qualitative
answers are sufficient (not quantitative).

The purpose of X-gal is to produce blue color.


The purpose of IPTG is to induce expression of beta galactosidase.
The purpose of Amp is to select plasmid carrying E. coli.

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BioC 4025W Fall 2017 Lab 7

Procedure 1

FIRST THING,if you did size exclusion, you would do an activity assay on your conc’t
SEC—Instead, do an activity assay on your conc’t AFC! This is also a good time to re-
assay any sample that you don’t have an activity assay data.

Protein Concentrations

Standard Curve

1. Set up a standard curve-Refer to Lab 2, Procedure 1:

a. Change wavelength on spec to 595 nm. Allow spec to equilibrate at that


wavelength.

b. Set up 3 ml cuvettes.

c. Set up standard curve along with your samples:

Reagent/Cuvette 1 2 3 4 5 6 7 8
BSA Standard, 1 mg/ml (µl) 0 10 20 30 40 50 75 100
Bradford reagent 3 ml →

d. Cuvette #1 is your blank (3 mL Bradford reagent, no BSA, no protein).


Blank with Bradford reagent.

Pour about 100 ml of BRADFORD REAGENT INTO THE 250 ml GLASS BEAKER at
your station.

NOTE: DO NOT put in plastic as it stains! Your plastic beakers have been
removed so they cannot be contaminated with Bradford Reagent. Put tips into the
large plastic beaker you share with the group across from you.

Reagent/Cuvette 1 2 3 4 5 6 7 8 etc.
Sample (µl) [see table next page]
Bradford reagent 3 ml →

Use 10 µl of your samples unless they are extremely “gunky” e.g. the crude sample.
You will need to dilute those samples 1 - 10 then use 10 µl of that diluted sample for
your assay. Be sure to account for the dilution in your calculations. Dilute with
water.
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BioC 4025W Fall 2017 Lab 7

2. Add 3 ml Bradford reagent to each cuvette and mix well immediately. Do not wait
unit you have added it to all cuvettes.

3. Incubate at least 10 minutes at RT. Once the color develops, it is stable for over
an hour. While it sits, now is a good time to peer evaluate LLR writing samples of
Methods and Results. Look at the Rubric Guidelines

4. Run NanoPhotometer at 595 wavelength.

5. Plot a Standard curve (y-axis = Abs., x-axis = µg protein)


Hint: you can use the ‘standard curve’ program on the nanophotometer.
a. Determine which part of curve is usable (linear).
b. Determine protein concentrations of your samples, the specific
activity, and the fold purification.
c. Complete the purification table:

Remember, this chart below will go into your large lab report.
Hint: you should have done an activity on the conc’t AFC sample.

Fraction Total Enzyme Total Recovery Protein Total Specific Fold


volume Activity units (%) (mg/mL) Protein activity purification
(mL) (U/mL) (U) (mg) (U/mg)
Crude (crude) 30 100 1.0
20,000 x g,
supernatant
(20,000xg)
65% dissolved pellet
(65% pellet)

Dialyzed 65% AS
pellet (dial 65%)

Pooled IEX fractions


(pool IEX)

Dialyzed IEX fractions


(dial IEX)

Pooled Affinity
fractions (pool AFC)
Conc’t Affinity fractions
(conc’t AFC)

Pooled Size Exclusion


fractions (Pool SEC)

Conc’t Size Exclusion


fractions (conc’t SEC)

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BioC 4025W Fall 2017 Lab 7

Procedure 2:

Results from Ligation/Transformation

Check the plates you streaked last time.

Circle two colonies from plate A or B or C and put your color tape by the circled
colonies. If you don’t have any colonies on either A or B, circle two colonies from plate
C. What probability would you expect? How could you estimate based on v:i DNA ratio?

If you don’t have colonies on A, B, or C, circle two colonies from someone else in the
lab. Put your initials on your color tape by these circles.

The colonies that you circled will be used to inoculate a 5 ml culture of LB/Amp so you
can do plasmid preps next week. You will get your cultures back next lab period.

INQUIRY: Design an
Just for thought: Awexperiment
snap! Doeswith
our the his-tag
plasmid using
have thechromatography.
B/W screen? How does
the protein bind and how would you get it off?
Based on vector:insert ratios and control plates, what is the probability that the
colony you pick will have your LDH gene insert cloned into the vector?

What are three or more ways we could test this to find out?

What would it take to repair this?

What’s Due Next Lab

Data and Summary/Conclusions Lab 7, Procedure 1

Lab Report 7 (there was no lab report 6 so it is 6-7), including data and questions.

Prelab write-up in notebook Lab 8, Procedures 1- 5, but pre-lab question is just to watch
the video below.
https://www.khanacademy.org/test-rep/mcat/biomolecules/enzyme-kinetics/v/an-introduction-to-enzyme-kinetics
Next lab 8 is during midterm week, so be sure to view Kahn academy enzyme kinetics
in advance.
CLEAN UP: SERIOUSLY, THIS IS AN EASY ONE….CHECK OUT THE HANDOUT,
EASY BREEZY.

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