Molecular Genetics 7

777120
Lab 2
Objectives
Understand the principles and applications of gel electrophoresis
Become proficient in the techniques of agarose gel electrophoresis
Construct standard curves and use experimental data to determine the sizes of
unknown DNA samples
Apply knowledge gained to generate a restriction map of the lamda virus genome
Introduction
!t is possile to determine the relative positions of a variety of restriction sites in a fragment of
DNA without sequencing it" #his process is called restriction mapping" Using the digests you set
up last week$ you will determine the relative positions of the Eco%!$ Hind!!! and Pst! sites in
DNA"
!nformation in your textook&
'amda () DNA pp *+,++
%estriction enzymes& pp -.,-+
%estriction mapping& pp -+,-/
0el electrophoresis& pp 11,12
Part A: Agarose Gel Electroporesis o! "estriction #igest
Materials
3ne45 mini agarose6 7"28 #B9 gel for each group of three
%estriction digests from 'a 4 : on ice
7"28 #B9 electrophoresis uffer$ .27 m' per gel tank for running gel
;ini agarose gel electrophoresis tanks
<ower supplies
7"28 #B9 electrophoresis uffer$ .27 m' per gel tank for running gel
=terile477m' measuring cylinders
=terile.27m' measuring cylinders
9thidium romide (47 mg6m')
=ample loading uffer$ 17 microfuge tues x 477'
;icrofuge test tues$ 4"2 l
;icrofuge test tue trays
=afety glasses
Hind!!! DNA in sample loading gel
3ne lunch ox per mini agarose gel
DNA Molecular Weight Marker II [Roche Applied Sciences # 10232!0001" #2!0 ng$%&'
Metod $In te sa%e groups o! tree as !or te restriction digest Lab 1&
>ou have een provided with one premade45 agarose gel for each group of three"
#he digests you set up last week were removed from 1+
o
C for you and stored at :.7
o
C i"e"
frozen" #hey have een removed from the freezer and are now on ice for you"
4" 0ently remove com from your agarose gel and cover the gel with .27m' of 7"2 8 #B9
gel electrophoresis uffer"
." Briefly spin your restriction digests in a microfuge (2 sec) (ensure tues are alanced) to
ring contents to the ottom of the tue"
3. <ipette 2' of sample loading uffer to each tue"
-" Briefly spin your tues again in a microfuge (2 sec) (ensure tues are alanced) to ring
contents to the ottom of the tue"
2" 'oad your gel as follows from left to right&
? @ _' @ ? _' Hind!!! (A +27ng)
lane .& 42 ' digest A
lane 1& 42 ' digest B
lane -& 42 ' digest C
lane 2& 42 ' digest D
lane *& 42 ' digest 9
lane +& 42 ' digest B
lane /& @ ? _' uncut DNA (A427ng)
*" %un gel at CCC D for CCC minutes" >our lecturer will show you how to start the gel
running" (Ehile your gel is running$ egin <art B)
+" 3nce the romophenol lue has run to F4cm from the end opposite the wells$ turn the
power supply off"
/" %emove gel from gel tank$ place into lunch ox and take to gel documentation camera"
G" #ake a photo of your gel"
Part ': "estriction Mapping
>ou will map the relative positions of the Eco%!$ Hind!!! and Pst! restriction sites in DNA"
1. Using a ruler$ measure H record the distance in mm that each of the DNA size markers
( Hind!!!) has migrated from the well"
." 3n semi,log graph paper$ plot a standard curve of this distance against the size of each
of the markers i"e" mark the distance (mm) on the x,axis (horizontal axis) and the ase
pair size on the y,axis (vertical axis)" <lot and connect these points in a est,fit straight
line"
1" ;easure H record the distances migrated for each of the ands in the other digests"
N3#9& you need to e consistent& measure from the ottom of the well to the ottom of the
and"
-" %efer to the graph y moving along the x,axis to the designated distanceI then move up
until you reach your est,fit line" Brom this point on the est,fit line$ find the
corresponding height on the ase pair size axis (y,axis)" %ead H record the size
measurement indicated on the ase pair size axis"

Part (: Anal)sis
Be prepared to answer these questions in the next la class" Ee will have a discussion aout
these questions and you will e called upon to contriute your answers and6or ideas to the
discussion"
4" >ou want to digest 4ug of 'amda DNA with 47U of the enzyme Eco%!" >ou have een
provided with the following&
47ug6l 'amda DNA
478 Eco%! uffer
47U6l Eco%!
100l sterile distilled water
Determine what volumes of each ingredient would e required to set up a reaction with a total
volume of .7l"
." 'amda DNA is a linear molecule of approximately 27k" Jow many Eco%! sites would
you predict there to e within this moleculeC Descrie how you calculated this"
1" Jow many DNA fragments would you therefore predict Eco%! to generate from 'amdaC
-" #he restriction site for Taq! is 2K,#C0A,1K" Jow many recognition sites for this enzyme
would you predict there to e in a 'amda moleculeC
2" Jow many DNA fragments would you therefore predict Taq! to generate from 'amdaC
Descrie how you calculated this"

*" Jow many fragments did Hind!!! produce y cutting 'amda DNA and what size were
theyC
Ingredient *olu%e
47ug6l 'amda DNA
478 Eco%! uffer
47U6l Eco%!
477l sterile distilled water
.7l total volume
+" Jow many times did this enzyme cut the DNAC
/" Jow many fragments of DNA did the Pst! restriction digest produceC
G" Jow many times did this enzyme cut the DNAC
47" Jow many fragments of DNA did the Eco%! restriction digest produceC
44" Jow many times did this enzyme cut the DNAC
4." Compare the lanes with your digests with each other" Do any of the lanes have ands in
commonC !f so$ which ands are theyC Ehat do they representC
41" Ehich lane has the smallest piece of DNAC Jow do you knowC
4-" Ehat does each of the ands on your gel representC
42" Ehat is the connection etween the restriction enzymes and these andsC
4*" Compare the pattern of fragments of each digest with the fragment pattern of the
molecular weight markers" Did any of your digests produce a similar patternC !f so$ which
oneC
4+" #o which electrode did the pieces of DNA moveC EhyC
4/" %ememer that the three samples of DNA started out the same" Assuming that each
sample was cut into fragments y the addition of three different restriction enzymes$ how
does a restriction digest give evidence that each enzyme cuts the DNA at different
locationsC
Gel Electroporesis
9lectrophoresis is the separation of charged molecules in an electric field" 9lectrophoresis is
used to separate protein or DNA molecules of different sizes"
Mobilit) o! #+A, "+A - Protein
#he moility of DNA is influenced y the fragmentsK molecular size and conformation" 'arger
molecules move more slowly than smaller fragments (Bigure 4)" =maller molecules move
through the gel matrix more readily than larger molecules$ so that molecules of different length$
such as restriction fragments$ separate"
Bigure 4" .eparation o! #+A !rag%ents o! di!!erent lengts b) gel electroporesis/
L .777 y E" J" Breeman and Company"
=uperhelical circular DNA moves faster than linear molecules$ which moves faster than nicked
circular DNA forms (Bigure .)"
Bigure .& Differences in moility oserved etween nicked circular$ supercoiled H linear DNA" #he relative positions
of the electrodes are indicated on the right (1)"
DNA and %NA molecules are highly charged near neutral pJ ecause the phosphate group in
each nucleotide contriutes one negative charge" As a result$ DNA and %NA molecules move
toward the positive electrode during gel electrophoresis (Bigure .)"
<roteins H %NA form extensive secondary structures" Denaturing electrophoresis is used to
reduce the impact of conformation on the migration of these molecules" Burther$ for proteins$
charge differences etween molecules is a complicating factor" #hus$ samples are treated to
have the same charge so that size can then ecome the most important factor when separating
proteins"
#he gel matrix restricts random diffusion of the moleculesI therefore$ molecules of different
length separate into MandsM whose width equals that of the well into which the original DNA
mixture was placed (Bigure 4)"
#he resolving power of gel electrophoresis is so great that single,stranded DNA molecules up to
aout 277 nucleotides long can e separated if they differ in length y only 4 nucleotide" #his
high resolution is critical to the DNA,sequencing procedures descried later" DNA molecules
composed of up to N.777 nucleotides usually are separated electrophoretically on
polyacrylamide gels$ and molecules from 277 nucleotides to .7 k on agarose gels"
Matrices
A gel is prepared y pouring a liquid containing either melted agarose or unpolymerised
acrylamide etween two glass plates a few millimeters apart" As the agarose solidifies or the
,ve
Ove
acrylamide polymerises into polyacrylamide$ a gel matrix forms consisting of long$ tangled
chains of polymers (Bigure 4)"
#he dimensions of the interconnecting channels$ or pores$ depend on the concentration of the
agarose or acrylamide used to form the gel" Because the pores are larger in agarose gels than
in polyacrylamide gels$ the former are used to separate large DNA fragments (N277 p to N.7
k) and the latter to separate small DNA fragments (4 nucleotide to N. k) and proteins"
<olyacrylamide gels are more hazardous H harder to use than agarose gels ut have very high
resolving power" Bor example$ polyacrylamide gels are used when sequencing DNA and for
separating proteins"
;oility is influenced y the porosity of the matrix" #he porosity of the matrix varies with its
concentration" #he migration of molecules is retarded y the porous gel matrix retards such that
smaller fragments can move faster than larger fragments" ;olecules are thus separated in the
matrix according to their relative size and conformation" =maller molecules are separated y
higher concentrations of the matrix while lower concentrations of the matrix allow separation of
larger molecules (#ale 4 H Bigure 1)"
#ale 4& A comparison of the ranges of separation of linear DNA for different agarose concentrations"
Amount of
Agarose in gel
(5w6v)
%ange of separation
of linear DNA (k)
7"1 2,*7
7"* 4,.7
7"+ 7"/,47
7"G 7"2,+
4". 7"-,*
4"2 7".,1
."7 7"4,.
Bigure 1& 9ffect of agarose concentration on the separation of DNA fragments" Agarose gels of 7"+5$ 4"75 H 4"25
are compared" #he same and in each DNA ample is indicated for each agarose concentration"
#he mixture of DNA fragments to e separated is layered in a well at the top of the gel and an
electric current is passed through the gel" DNA fragments move toward the positive pole at a
rate inversely proportional to the log of their length$ forming ands that can e visualized y
autoradiography (if the fragments are radiolaelled) or y addition of a fluorescent dye such as
ethidium romide (Bigures . and 1)"
#enaturing gels
%NA and proteins form secondary structures (they fold on themselves)" #hese types of
molecules need to e denatured (i"e" unfolded) so that their size and not their conformation
influences their rate of migration" Denaturing gels allow this"
%NA molecules are denatured with formaldehyde H formamide efore eing applied to an
agarose matrix containing formaldehyde" #hese agents cause the %NA molecules to ecome
linear$ thus allowing separation according to size and reducing the influence of conformation"
#o create denaturing gels for proteins$ the sodium dodecyl sulphate (=D=) and the disulfide
reducing agent$ mercaptoethanol$ are added to the polyacrylamide matrix" =D= is a detergent
which converts all proteins into linear structures coated in negatively charged =D="
;ercaptoethanol assists the denaturing process y reducing all disulphide onds"
Denaturing polyacrylamide gels containing urea H formamide are used for separating DNA
fragments within a sequencing gel" #his prevents the nucleic acids from renaturing$ improving
the resolving power of this gel system to allow separation of DNA fragments differing in length
y one nucleotide" Eithout this denaturation$ it would e impossile to read a DNA sequence"
"unning a gel
!n order to create an electric field$ the gel matrix must e ale to conduct electricity" #he matrix
alone cannot do this effectively" #hus$ the matrix is mixed with a salt solution that does conduct
electricity efficiently" #he gel matrix contains this salt mixture and is sumerged in a uffer that
is the same salt concentration as is present within the gel" #he uffer also controls the pJ of the
solution during electrophoresis"
'oading dye is added to the samples prior to electrophoresis and serves to weight the samples$
allowing them to sink to the ottom of the wells" #he dye also provides a visual marker to show
the progress of the electrophoresis"
#he intercalating dye ethidium romide is either mixed in with the gel at the time of preparation
or is used as a stain after electrophoresis" 9thidium romide allows DNA H %NA to e detected
y visile fluorescence when illuminated with ultraviolet light" <roteins are stained with a stain
such as Coomassie Blue or =ypro %uy"
Bigure -& #he structure of ethidium romide"
9thidium romide is a planar molecule that inds to DNA y intercalating etween the ase
pairs" Binding concentrates ethidium in the DNA and also increases its intrinsic
fluorescence" As a result$ when the gel is illuminated with ultraviolet light$ the regions of
the gel containing DNA fluoresce much more rightly than the regions of the gel without
DNA (Bigure 2)"
Molecular 0eigt standards
;olecular weight standards are also run on the gel (Bigure 2)" #hese are to assist with
estimating the size of nucleic acid molecules of unknown length y comparing the
migration with that of molecules of known length"
DNA fragments move toward the positive pole at a rate inversely proportional to the log
47
of their
length or molecular weight" ;olecules of the same size migrate through a gel at the
same rate as each other"
!f you plot the distance from the well that the molecules have migrated against the log
47
of either
their molecular weights or numer of ase pairs$ a roughly straight line will appear" #herefore$
we can estimate the molecular weight or size of a DNA fragment when compared with
molecules of known size run on the same gel i"e" molecular weight standards"
Bigure 2& An agarose gel of DNA fragments from different samples" #he DNA at the top end of the gel is larger than
DNA at the ottom end" #he lanes on each end (left H right) contain DNA molecular weight markers i"e" DNA
fragments of known size" #hese are used to determine the sizes of the fragments in the samples contained in the
lanes in etween" #he DNA has een stained with ethidium romide and then$ following electrophoresis$ visualised
under UD light"
"estriction Mapping
%estriction mapping is used to determine the relative position of different restriction enzymes
sites within a DNA sequence" #he different DNA fragments generated with different restriction
enzymes can e used to do this" Digesting DNA with single restriction enzymes doesnKt give us
the information we need in order to create a restriction mapI we need to do doule digests i"e"
digesting the DNA with two enzymes at once"
Bor example$ in Bigure * a DNA sample has een digested with Eco%!$ Hind!!! and the two
enzymes together" !t can e seen which fragments generated y one enzyme have een cut y
the other enzyme" Eith this knowledge$ the relative positions of the two restriction enzyme sites
can e determined (Bigure +)
Bigure *& Digests of a DNA sample with the restriction enzymes Eco%! and Hind!!! on their own or together" #he
molecular weight markers are in the lane laelled P;Q and their sizes in ase pairs given on the right hand side"
Bigure +& #he relative positions of the Eco%! and Hind!!! sites for the DNA sample digested in Bigure *"
427 p
-77 p
4127 p
4777 p
*77 p
277 p
Eco%! sites
Hind!!! sites
.777 p
4277
4777
G77
/77
+77
*77
277
-77
177
.77
477
Eco%!
Hind!!!
Eco%! +
Hind!!!
;
"e!erences:
4" http&66www"nci"nlm"nih"gov6entrez6query"fcgiC
cmdA=earchHdAooksHdoptcmdlA0enBookJ'HtermAelectrophoresisOANDOmc
52Book52DOANDO4722*.52Buid52DHridAmc"section"4*1+R4*-/" Downloaded
.77-"
." http&66oceanexplorer"noaa"gov6explorations671io6logs6sept476media6lasonolide1"html "
Downloaded .77-"
3. http&66arl"cvms"colostate"edu6hooks6genetics6iotech6gels6agardna"html " Downloaded
.77-"
Preparation +otes
Prior to Lab 2
Ensure te !ollo0ing is in stoc1 !or Part A 2 order 0ell aead o! ti%e:
1 tues DNA ;olecular Eeight ;arker !! S%oche Applied =ciences R 47.1*.27774T
(.27 ng6m')" Note& this is ? _*__ Hind!!!"
o =tore at :/7
o
C
Ensure te !ollo0ing is available !or Part A:
47 x 45 mini agarose6 7"28 #B9 gel for each group of three already poured with / well
coms" 'eave coms in gel for students to remove"
o A ulk mixture of agarose can e made up
o Bor each small gel&
=et up gel tray in gel rig with / well com in gel tray"
#ransfer 7"*g agarose to a flask$ add *7mls 7"28 #B9
;elt agarose in microwave oven
Allow the agarose to cool down to a temperature at which you can hold the
flask reasonaly comfortaly in your hands"
Add 7"*' 47mg6m' ethidium romide to cooled agaroseI swirl gently to
mix"
(A34IO+: Ethidium bromide is a mutagen. Ensure you are wearing
gloves and safety glasses when dispensing ethidium bromide and
pouring your gel.
<our gel mixture into assemled gel rig6tray in one movementI remove any
air ules
'eave gel to set"
(A34IO+: To avoid creating bubbles or ripples, do not touch gel rig/tray
while gel is setting.
3nce gel has set leave coms in for students to remove"
%estriction digests from 'a 4 : remove from freezer and place on ice"
7"28 #B9 electrophoresis uffer$ 47 x .27 m'
47 x ;ini agarose gel electrophoresis tanks
* x <ower supplies
47 x =terile477m' measuring cylinders
47 x =terile .27m' measuring cylinders (if 7"28 #B9 electrophoresis uffer not provided
in .27 ml aliquots)
9thidium romide (47 mg6m')& 47 aliquots of 477'
=ample loading uffer$ 47 aliquots x 477'
;icrofuge test tues$ 4"2 l& 2 x 4' eakers full$ sterilised
47 x ;icrofuge test tue trays
=afety glasses& one pair for each student"
47 x lunch oxes for mini agarose gels
Uncut DNA in sample loading uffer"
o #ransfer 47' of .27ng6' DNA to a fresh tue"
o Add 4G7 ' sterile distilled water and 27 ' sterile sample loading dye"
o ;ix well y pipetting up H down"
o Dispense into 27 ' aliquots and lael as Puncut 47ng6'Q
o =tore aliquots at :.7
o
C and place on ice prior to class"
Hind!!! DNA in sample loading gel" Dilute . tues of DNA ;olecular Eeight ;arker !!
S%oche Applied =ciences R 47.1*.27774T (.27 ng6') as follows&
o #ransfer 477' of .27ng6' DNA to a fresh tue"
o Add 127 ' sterile distilled water and 27 ' sterile sample loading dye"
o ;ix well y pipetting up H down"
o Dispense into 27 ' aliquots and lael as P Hind!!! 27ng6'Q
o =tore aliquots at :.7
o
C and place on ice prior to class"
Ensure te !ollo0ing is available !or Part ':
=emi log raph paper
A!ter Lab 2:
Nothing to do"