777120 2006 Lab 2 Student Version

8/13/2019 777120 2006 Lab 2 Student Version

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Molecular Genetics 7777120

Lab 2Objectives

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 ofunknown 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 ofDNA 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

Materials3ne45 mini agarose6 7"28 #B9 gel for each group of three

%estriction digests from 'a 4 : on ice7"28 #B9 electrophoresis uffer$ .27 m' per gel tank for running gel;ini agarose gel electrophoresis tanks


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." Briefly spin your restriction digests in a microfuge (2 sec) (ensure tues are alanced) toring contents to the ottom of the tue"

3. our lecturer will show you how to start the gelrunning" (Ehile your gel is running$ egin


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Part (: Anal)sis

Be prepared to answer these questions in the next la class" Ee will have a discussion aoutthese questions and you will e called upon to contriute your answers and6or ideas to thediscussion"

4" >ou want to digest 4ug of 'amda DNA with 47U of the enzyme Eco%!" >ou have eenprovided 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 molecule Descrie how you calculated this"

1" Jow many DNA fragments would you therefore predict Eco%! to generate from 'amda

-" #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 molecule

2" Jow many DNA fragments would you therefore predict Taq! to generate from 'amda

Descrie how you calculated this"

*" Jow many fragments did Hind!!! produce y cutting 'amda DNA and what size were

they

Ingredient *olu%e

47ug6l 'amda DNA

478 Eco%! uffer

47U6l Eco%!

477l sterile distilled water

.7l total volume


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+" Jow many times did this enzyme cut the DNA

/" Jow many fragments of DNA did the Pst! restriction digest produce

G" Jow many times did this enzyme cut the DNA

47"Jow many fragments of DNA did the Eco%! restriction digest produce

44"Jow many times did this enzyme cut the DNA

4."Compare the lanes with your digests with each other" Do any of the lanes have ands in

common !f so$ which ands are they Ehat do they represent

41"Ehich lane has the smallest piece of DNA Jow do you know

4-"Ehat does each of the ands on your gel represent

42"Ehat is the connection etween the restriction enzymes and these ands

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 pattern !f so$ which

one

4+"#o which electrode did the pieces of DNA move Ehy


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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

locations


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Gel Electroporesis9lectrophoresis is the separation of charged molecules in an electric field" 9lectrophoresis isused 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" 'argermolecules move more slowly than smaller fragments (igure 4)" =maller molecules movethrough the gel matrix more readily than larger molecules$ so that molecules of different length$such as restriction fragments$ separate"


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igure 4" .eparation o! #+A !rag%ents o! di!!erent lengts b) gel electroporesis/L .777 y E" J" reeman and Company"

=uperhelical circular DNA moves faster than linear molecules$ which moves faster than nickedcircular DNA forms (igure .)"

igure .& Differences in moility oserved etween nicked circular$ supercoiled H linear DNA" #he relative positionsof the electrodes are indicated on the right (1)"

DNA and %NA molecules are highly charged near neutral pJ ecause the phosphate group ineach nucleotide contriutes one negative charge" As a result$ DNA and %NA molecules movetoward the positive electrode during gel electrophoresis (igure .)"


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acrylamide polymerises into polyacrylamide$ a gel matrix forms consisting of long$ tangledchains of polymers (igure 4)"

#he dimensions of the interconnecting channels$ or pores$ depend on the concentration of theagarose or acrylamide used to form the gel" Because the pores are larger in agarose gels thanin polyacrylamide gels$ the former are used to separate large DNA fragments (277 p to .7k) and the latter to separate small DNA fragments (4 nucleotide to . k) and proteins"


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#enaturing gels%NA and proteins form secondary structures (they fold on themselves)" #hese types ofmolecules need to e denatured (i"e" unfolded) so that their size and not their conformationinfluences 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 ecomelinear$ 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 disulfidereducing agent$ mercaptoethanol$ are added to the polyacrylamide matrix" =D= is a detergentwhich 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 DNAfragments within a sequencing gel" #his prevents the nucleic acids from renaturing$ improvingthe 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 matrixalone cannot do this effectively" #hus$ the matrix is mixed with a salt solution that does conductelectricity efficiently" #he gel matrix contains this salt mixture and is sumerged in a uffer thatis the same salt concentration as is present within the gel" #he uffer also controls the pJ of thesolution 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 preparationor is used as a stain after electrophoresis" 9thidium romide allows DNA H %NA to e detectedy visile fluorescence when illuminated with ultraviolet light"


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9thidium romide is a planar molecule that inds to DNA y intercalating etween the asepairs" Binding concentrates ethidium in the DNA and also increases its intrinsicfluorescence" As a result$ when the gel is illuminated with ultraviolet light$ the regions ofthe gel containing DNA fluoresce much more rightly than the regions of the gel withoutDNA (igure 2)"

Molecular eigt standards

;olecular weight standards are also run on the gel (igure 2)" #hese are to assist withestimating the size of nucleic acid molecules of unknown length y comparing themigration with that of molecules of known length"

DNA fragments move toward the positive pole at a rate inversely proportional to the log47of theirlength or molecular weight" ;olecules of the same size migrate through a gel at thesame rate as each other"

!f you plot the distance from the well that the molecules have migrated against the log47of eithertheir 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"

igure 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" DNAfragments 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$ visualisedunder U light"


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"estriction Mapping%estriction mapping is used to determine the relative position of different restriction enzymessites within a DNA sequence" #he different DNA fragments generated with different restrictionenzymes can e used to do this" Digesting DNA with single restriction enzymes doesnKt give usthe 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"

or example$ in igure * a DNA sample has een digested with Eco%!$ Hind!!! and the twoenzymes together" !t can e seen which fragments generated y one enzyme have een cut ythe other enzyme" Eith this knowledge$ the relative positions of the two restriction enzyme sitescan e determined (igure +)

igure *& Digests of a DNA sample with the restriction enzymes Eco%! and Hind!!! on their own or together" #hemolecular weight markers are in the lane laelled P;Q and their sizes in ase pairs given on the right hand side"

igure +& #he relative positions of the Eco%! and Hind!!! sites for the DNA sample digested in igure *"

427 p

-77 p

4127 p

4777 p*77 p

277 p

Eco%! sites

Hind!!! sites

.777 p42774777G77/77+77*77277

-77177

.77

477

Eco%! Hind!!! Eco%!+Hind!!!

;


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"e!erences:4" http&66www"nci"nlm"nih"gov6entrez6query"fcgi

cmd=earchHdooksHdoptcmdl0enBookJ'HtermelectrophoresisOANDOmc52Book52DOANDO4722*.52Buid52DHridmc"section"4*1+R4*-/" Downloaded.77-"

." http&66oceanexplorer"noaa"gov6explorations671io6logs6sept476media6lasonolide1"html"Downloaded .77-"

3. http&66arl"cvms"colostate"edu6hooks6genetics6iotech6gels6agardna"html"Downloaded.77-"
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://oceanexplorer.noaa.gov/explorations/03bio/logs/sept10/media/lasonolide3.htmlhttp://oceanexplorer.noaa.gov/explorations/03bio/logs/sept10/media/lasonolide3.htmlhttp://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.htmlhttp://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.htmlhttp://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.htmlhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=electrophoresis+AND+mcb%5Bbook%5D+AND+105562%5Buid%5D&rid=mcb.section.1637#1648http://oceanexplorer.noaa.gov/explorations/03bio/logs/sept10/media/lasonolide3.htmlhttp://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html


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Preparation +otes

Prior to Lab 2

Ensure te !olloing is in stoc !or Part A order ell 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 :/7oC

Ensure te !olloing 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 or 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 wearinggloves and safety glasses when dispensing ethidium bromide andpouring your gel.


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o Dispense into 27 ' aliquots and lael as Puncut 47ng6'Q

o =tore aliquots at :.7oC 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 PHind!!! 27ng6'Q

o =tore aliquots at :.7oC and place on ice prior to class"

Ensure te !olloing is available !or Part ':

=emi log raph paper

A!ter Lab 2:

Nothing to do"

777120 2006 Lab 2 Student Version

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