DNA Protocols & Applications

9/12/2016
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ConsiderationsforisolationandquantificationofbothgenomicDNAandplasmidDNA
Thissectiondescribesconsiderationsforisolationandquantificationofboth
genomicDNAfromdifferentsamplesourcesandplasmidDNA.Italsodealswith
commonplasmidDNAprocedures,includinghowtomakeandtransform
competentcells,howtocultureandhandleplasmidcontainingcells,and
commonlyusedtechniquesforanalysisofgenomicDNA.
Epigenetics
Transfection
Protein
AnimalCellCulture
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WhatisDNA?
DNAextractiontechnologies
WorkingwithDNA:Goodlaboratorypractice
Conversionsfornucleicacids:DNA
SpectrophotometricmeasurementofDNAconcentration
SamplestoragepriortoextractionofgenomicDNA
SampledisruptionforextractionofgenomicDNA
WorkingwithDNA:Goodmicrobiologicalpractice
StorageofE.colistrains
Plasmidspecifications
Bacterialcultivationmediaandantibiotics
Lysisofbacterialcellsforplasmidpurification
TransformationofDNA
TransformationofcompetentE.coli
IsopropanolprecipitationofDNA
StorageofDNA
Endotoxinsandwhattoconsider
QuantificationofDNA
Spectrophotometry
Fluorometry
Agarosegel
RestrictionendonucleasedigestionofDNA
LigationofDNA
DNAanalysisusinganalyticalgels
Pouringanagarosegel
Runninganagarosegel
Visualanalysisofthegel
AnalysisofDNAbySouthernblotting
DNAcleanup
References
WhatisDNA?
GenomicDNA
GenomicDNAconstitutesthetotalgeneticinformationofanorganism.ThegenomesofalmostallorganismsareDNA,the
onlyexceptionsbeingsomevirusesthathaveRNAgenomes.GenomicDNAmoleculesaregenerallylarge,andinmost
organismsareorganizedintoDNAproteincomplexescalledchromosomes.Thesize,numberofchromosomes,and
natureofgenomicDNAvariesbetweendifferentorganisms(seetable Sizesandmolecularweightsofvariousgenomic
DNAs).ViralDNAgenomesarerelativelysmallandcanbesingleordoublestranded,linear,orcircular.Allother
organismshavedoublestrandedDNAgenomes.Bacteriahaveasingle,circularchromosome.Ineukaryotes,most
genomicDNAislocatedwithinthenucleus(nuclearDNA)asmultiplelinearchromosomesofdifferentsizes.Eukaryotic
cellsadditionallycontaingenomicDNAinthemitochondriaand,inplantsandlowereukaryotes,thechloroplasts.ThisDNA
isusuallyacircularmoleculeandispresentasmultiplecopieswithintheseorganelles.
SizesandmolecularweightsofvariousgenomicDNAs
Organism
Basepairsperhaploid
genome
Molecularweightofgenome
(daltons)
Numberof
chromosomes
SV40
5243
3.4x106
F174
5386
3.5x106
Adenovirus2
35,937
2.3x107
https://www.qiagen.com/us/resources/molecularbiologymethods/dna/
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Lambda
48,502
3.2x107
Escherichiacoli
4.7x106
3.1x109
x=1
Saccharomyces
1.5x107
9.8x109
2x=32
Dictyostelium
discoideum
5.4x107
3.5x1010
x=6
Arabidopsisthaliana
7.0x107
4.6x1010
2x=10
Caenorhabditiselegans
8.0x107
5.2x1010
2x=12
1.4x108
9.1x1010
2x=8
1.2x109
7.8x1011
2x=78
Musmusculus(mouse)
2.7x109
1.8x1012
2x=40
Rattusnorvegicus(rat)
3.0x109
2.0x1012
2x=42
Xenopuslaevis
3.1x109
2.0x1012
2x=36
Homosapiens
3.3x109
2.1x1012
2x=46
Zeamays
3.9x109
2.5x1012
2x=20
Nicotianatabacum
4.8x109
3.1x1012
2x=48
cerevisiae
Drosophila
melanogaster
Gallusdomesticus
(chicken)
Adaptedfromreferences1and2.
GenomicDNAcontainsgenes,discreteregionsthatencodeaproteinorRNA.AgenecomprisesthecodingDNA
sequence,aswellastheassociatedregulatoryelementsthatcontrolgeneexpression.Nucleareukaryoticgenesalso
containnoncodingregionscalledintrons.Thenumberofgenesvarieswidelybetweendifferentorganisms.CodingDNA
representsonlyasmallfractionofeukaryoticgenomicDNA:thebulkoftheDNAisnoncoding,muchofwhichismadeupof
repetitivesequences.SomenoncodingDNAhasstructuralandregulatoryfunctionshowever,thefunctionofmostofthis
DNAislargelyunknown.Thenumberofcopiesofeachgeneticlocuspresentinacell,orploidy,alsovariesbetween
organisms.Thesomatic(body)cellsoforganismsthatreproducesexuallyareusuallydiploid,havingtwosetsof
homologouschromosomesandhencetwocopiesofeachgeneticlocus,whilethegerm(reproductive)cellsarehaploid
andhaveonlyonecopyofeachchromosome.Prokaryoticcellsarehaploid.Someplantsarepolyploid,forexample,
modernwheat,whichishexaploid(sixcopiesofeachchromosome).
PlasmidDNA
BacterialplasmidsareclosedcircularmoleculesofdoublestrandedDNAthatrangeinsizefrom1to>200kb.Theyare
foundinavarietyofbacterialspecies,wheretheybehaveasadditionalgeneticunitsinheritedandreplicated
independentlyofthebacterialchromosome.However,theyrelyuponenzymesandproteinsprovidedbythehostfortheir
successfultranscriptionandreplication.
Plasmidsoftencontaingenesthatcodeforenzymesthatcanbeadvantageoustothehostcellinsomecircumstances.The
encodedenzymesmaybeinvolvedinresistanceto,orproductionof,antibiotics,resistancetotoxinsfoundinthe
environment(e.g.,complexorganiccompounds),ortheproductionoftoxinsbythebacteriaitself.
Oncepurified,plasmidDNAcanbeusedinawidevarietyofdownstreamapplicationssuchassequencing,PCR,
expressionofproteins,transfection,andgenetherapy.
DNAextractiontechnologies
Backtotop
DNAcanbepurifiedusingmanydifferentmethodsandthedownstreamapplicationdetermineshowpuretheDNAshould
be.Inadditiontoisolationusinghomemademethods(e.g.,CsClgradients),DNAextractionkitsareavailablefrommany
suppliers.Thecharacteristicsofthe3mostcommontypesofDNAextractionkitareshowninthetable Characteristicsof
commonDNAextractionkits.
CharacteristicsofcommonDNAextractiontechnologies
Anionexchange
Silicamembranetechnology
Magneticparticletechnology
Whatitis
Solidphase,anionexchange
chromatography
Selectiveadsorptiontosilica
membranes
Bindingtomagneticsilica
particlesundercontrolledionic
conditions
Binding:variablesaltandpH
Elution:variablesaltandpH
Binding:highsalt
Elution:lowsalt
Binding:highsalt
Elution:lowsalt
Readytouseeluate
Delivershighpuritynucleic
acidsforuseinmost
Readytouseeluate
Delivershighpuritynucleic
acidsforuseinmost
Procedure
Alcoholprecipitation
Deliversultrapure,transfection
Advantages gradeDNAforoptimalresultsin
sensitiveapplications
downstreamapplications
Fast,inexpensive
downstreamapplications
Fast,inexpensive
Nosilicaslurrycarryover,no
alcoholprecipitation
Easytoautomatenoalcohol
precipitation
AnionexchangemethodsyieldDNAofapurityandbiologicalactivityequivalenttoatleasttworoundsofpurificationin
CsClgradients,inafractionofthetime.Purifiednucleicacidsareofthehighestpossiblequalityandareidealforsensitive
downstreambiologicalapplications,suchastransfection,microinjection,sequencing,andgenetherapyresearch.
Silicamembranetechnologyyieldshighpuritynucleicacidssuitableformostmolecularbiologyandclinicalresearch
applications,suchasrestrictiondigestion,ligation,labeling,amplification,andradioactiveandfluorescentsequencing.
Magneticparticletechnologyyieldshighpuritynucleicacidssuitableformostmolecularbiologyapplicationsusedin
clinicalandveterinaryresearch,suchasrestrictiondigestion,ligation,labeling,amplification,andradioactiveand
fluorescentsequencing.Magneticparticletechnologycanoftenbeautomatedtoenablefastandeconomicalnucleicacid
purificationprocedures.
WorkingwithDNA:Goodlaboratorypractice
Backtotop
HandlingDNA
DNAisarelativelystablemolecule.However,introductionofnucleasestoDNAsolutionsshouldbeavoidedasthese
enzymeswilldegradeDNA.GenomicDNAconsistsofverylargeDNAmolecules,whicharefragileandcanbreakeasily.
ToensuretheintegrityofgenomicDNA,excessiveandroughpipettingandvortexingshouldbeavoided.DNAissubjectto
acidhydrolysiswhenstoredinwater,andshouldthereforebestoredinTEbuffer,seetable TEbuffer,pH7.4.
TEBuffer,pH7.4
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TEBuffer,pH7.4
Component
Volume
1MTrisCl,pH7.4
10ml
0.5MEDTA,pH8.0
2ml
Conversionsfornucleicacids:DNA
Backtotop
MolecularweightconversionsforDNA
MWofadoublestrandedDNAmolecule(sodiumsalt)=(numberofbasepairs)x(662daltons/basepair)
MWofasinglestrandedDNAmolecule(sodiumsalt)=(numberofbasepairs)x(331daltons/basepair)
MWofaDNAoligonucleotide(sodiumsalt,pH7):
MW=(NAx335.2)+(NCx311.2)+(NCx351.2)+(NTx326.2)+P
WhereNX=thenumberofresiduesoftherespectivenucleotidewithintheoligonucleotide(theMWlistedforeach
nucleotideistheMWofthatnucleotide,withassociatedsodium,incorporatedintheoligonucleotide)
Fordephosphorylatedoligonucleotides:P=84.0
Forphosphorylatedoligonucleotides:P=40.0
MolecularconversionsforDNA
MolarconversionsforDNAcanbefoundinthetables MicrogramDNAconversionsand PicomoleDNAconversions.
Protein/DNAconversionscanbefoundinthetable Protein/DNAconversions.
MicrogramDNAconversions
1g
pmol
Molecules
20boligonucleotide
152
9.1x1013
1000bpDNA
1.52
9.1x1011
pUC19DNA(2686bp)
0.57
3.4x1011
pBR322DNA(4363bp)
0.35
2.1x1011
LambdaDNA(48,502bp)
0.03
1.8x1010
PicomoleDNAconversions
1pmol
Micrograms
20boligonucleotide
0.0066
1000bpDNA
pUC19DNA(2686bp)
0.66
1.77
pBR322DNA(4363bp)
LambdaDNA(48,502bp)
2.88
32.01
Protein/DNAconversions
1pmol
DNA
10,000Da
30,000Da
270bp
810bp
100,000Da
2.7kb
1kbofDNAencodes333aminoacids@3.7x104Daltons.
SpectrophotometricmeasurementofDNAconcentration
Backtotop
TheconcentrationofDNAandRNAshouldbedeterminedbymeasuringtheabsorbanceat260nm(A260 )ina
spectrophotometer.Foraccuracy,absorbancereadingsat260nmshouldfallbetween0.15and1.0.
PureDNAhasanA260 /A280 ratioof1.82.0in10mMTrisCl,pH8.5.
StrongabsorbanceatA280 resultinginalowA260 /A280ratioindicatesthepresenceofcontaminants,suchasproteins.
Strongabsorbanceat270nmand275nmmayindicatethepresenceofcontaminatingphenol.
Absorbanceat325nmsuggestscontaminationbyparticulatesinthesolutionordirtycuvettes.
Spectrophotometricconversionsfromabsorbanceat260nm
1A260 unit
Concentration(g/ml)*
dsDNA
ssDNA
50
33
Oligonucleotides
2030
*ThisrelationshipisonlyvalidformeasurementsmadeatneutralpH,andisbasedonastandard1cmpath.
Adaptedfromreference1.
SamplestoragepriortoextractionofgenomicDNA
Backtotop
ThequalityofthestartingmaterialaffectsthequalityandyieldoftheisolatedDNA.ThehighestDNAyieldandqualityis
achievedbypurifyinggenomicDNAfromfreshlyharvestedtissuesandcells.Ifsamplescannotbeprocessedimmediately
afterharvesting,theyshouldbestoredunderconditionsthatpreserveDNAintegrity.Ingeneral,genomicDNAyieldswill
decreaseifsamples,particularlyanimalsamples,arestoredateither28Cor20Cwithoutprevioustreatment.In
addition,repeatedfreezingandthawingoffrozensamplesshouldbeavoidedasthiswillleadtogenomicDNAofreduced
sizeortoreducedyieldsofpathogenDNA(e.g.,viralDNA).Recommendationsforstorageofdifferentstartingmaterialsare
discussedbelow.
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Blood
Ananticoagulantshouldbeaddedtobloodsamplesthatwillbestored.Forexample,bloodsamplestreatedwithheparinor
EDTAcanbestoredat28Cforafewdaysorat20Cor80Cforafewweeks.Alternatively,bloodsamplescanbe
treatedwithACDSolutionB(0.48%citricacid,1.32%sodiumcitrate,1.47%glucoseuse1mlper6mlblood)andstored
foratleast5daysat28Cor1monthat20C.Forlongtermstorage,bloodnucleicanbepreparedandstoredat20C.
Otherclinicalsamples
Mostbiologicalfluids(e.g.,plasma,serum,andurine)andstoolsamplescanbestoredat28Cforseveralhours.
Freezingat20Cor80Cisrecommendedforlongtermstorage.Swabscanbestoreddryatroomtemperature.
Formalinfixationandparaffinembedding(FFPE)isanothermeansofsamplestorageandisparticularlyrelevantforclinical
tissuesamples.Dependingonthetissuetype,thespeedatwhichbiomoleculesaredegraded,induced,ormodified
followingharvestingcanvary.Therefore,theproceduresfortissueremovalandfixationshouldbedoneasquicklyas
possible.
Fixationoftissuesinvolvesplacingspecimensinaformalinsolution,whichcanvaryincomposition(atypical10%formalin
solutionmaycontain3.7%formaldehydeaswellas11.5%methanol).Theresultingchemicalreactionleadstocrosslinks
betweenbiomolecules,includingcrosslinksbetweennucleicacids,betweenproteins,andbetweennucleicacidsand
proteins.Foroptimalresults,neutralbufferedformalinsolutionshouldbeusedinsteadofunbufferedoracidicformalin
solutions.Neutralbufferslowsdownthedegradationofformalin,whosedegradationproductsarebelievedtocontributeto
impairingnucleicacidquality.
Theratioofformalintotissueshouldbeatleast10:1toensureoptimalfixation.Thisiseasytoachievewhenworkingwith
smalltissuespecimens,suchasneedlebiopsies.However,whendealingwithlargetissuesamplestheremaybe
insufficientformalinforfixation.Inthiscase,sectionsofthetissueshouldbecutforformalinfixation.Tissuesshouldbefixed
fornomorethan24hourstoavoidoverfixation.
Afterfixationinformalin,tissuespecimensareembeddedinparaffin,aprocesswhichconsistsofseveralsteps.Thefirst
stepisdehydration,wherewaterisreplacedbyanalcohol,usuallyethanol.Thisisfollowedbyclearing,wherethealcohol
isreplacedbyxyleneoraxylenesubstitute,andbyimpregnation,wherexyleneisreplacedbyparaffin.Thefinalstepis
embedding,wheretheentirespecimenissurroundedwithparaffin.Itisimportantthattissuespecimensarefully
dehydratedpriortoimpregnation,asresidualwatermayleadtosampledegradation.Werecommendalwaysusingfresh
alcoholandxylene,toavoidanypossibilityofcarryoverofwaterfromprevioususes.Toensureoptimalrecoveryofusable
DNAfromFFPEsamples,lowmeltingtemperatureparaffinshouldbeusedinstead.Inaddition,paraffincontaining
additivessuchasbeeswaxshouldbeavoided,astheymayinterferewithrecoveryofbiomolecules.
Animaltissue
Freshlyharvestedtissuecanbeimmediatelyfrozenandstoredat20C,80C,orinliquidnitrogen.Lysedtissuesamples
canbestoredinasuitablelysisbufferforseveralmonthsatambienttemperature.
Animalandhumantissuescanalsobefixedforstorage.Werecommendusingfixativessuchasalcoholandformalin
however,longtermstorageoftissuesinformalinwillresultinchemicalmodificationoftheDNA.Fixativesthatcausecross
linking,suchasosmicacid,arenotrecommendedifDNAwillbeisolatedfromthetissue.ItisalsopossibletoisolateDNA
fromparaffinembeddedtissue(see Otherclinicalsamples).
Animal,yeast,andbacterialcellcultures
Centrifugeharvestedcellcultures,removethesupernatant,andthenstorethecellsat20Cor80C.Alternatively,
animalcellnucleicanbepreparedandstoredat20C.
Planttissue
Freshleavesandneedlesfrommostplantspeciescanbestoredforupto24hoursat4CwithoutaffectingDNAqualityor
yield.Ingeneral,samplesthatwillbestoredforlongerthan24hoursshouldbestoredat80C.However,somesamples
(e.g.,treebuds)canbestoredforseveraldaysat4C.Tissuesstoredat4Cshouldbekeptinaclosedcontainertoprevent
dehydration.Largesamples(e.g.,branches)canbestoredinaplasticbagcontainingawetpapertowel.
Ifitisnotpracticaltostorefrozensamples,anumberofmethodsareavailablefordryingplanttissue,forexample,silicagel,
fooddehydrators,orlyophilizers(3).TopreventDNAdegradation,materialshouldbecompletelydesiccatedinlessthan24
hours.Driedsamplesshouldbekeptinthedarkatroomtemperatureunderdesiccatingorhermeticconditionsforlong
termstorage.Dependingonhowthesamplewashandled,theDNAinherbariumandforensicsamplesmaybedegraded.
Disruptedplantmaterialcanbestoredinasuitablelysisbufferforseveralmonthsatambienttemperature.
Fungalmaterial
Myceliumshouldbeharvesteddirectlyfromaculturedishorliquidculture.Forliquidcultures,thecellsshouldbepelleted
bycentrifugationandthesupernatantremovedbeforeDNAisolationorstorage.Harvestedsamplescanbeeitherdirectly
frozenorfreezedried,andstoredat80C.
SampledisruptionforextractionofgenomicDNA
Backtotop
Completedisruptionandlysisofcellwallsandplasmamembranesofcellsandorganellesisanabsoluterequirementfor
allgenomicDNAisolationprocedures.Incompletedisruptionresultsinsignificantlyreducedyields.
Disruptionmethods
Lysisbuffer
Disruptiongenerallyinvolvesuseofalysisbufferthatcontainsadetergent(forbreakingdowncellularmembranes)anda
protease(fordigestionofproteincellularcomponents).Thechoiceofproteasedependsonthelysisbufferused.Some
sampletypesrequireadditionaltreatmentforefficientlysisthisisdescribedinmoredetailin Specialconsiderationsfor
isolatinggenomicDNAfromdifferentsamplesources.
Disruptionusingrotorstatorhomogenizers
Rotorstatorhomogenizersthoroughlydisruptanimalandplanttissuesin590secondsdependingonthetoughnessof
thesample.Therotorturnsatveryhighspeedcausingthesampletobedisruptedbyacombinationofturbulenceand
mechanicalshearing.Foamingofthesampleshouldbekepttoaminimumbyusingproperlysizedvessels,bykeepingthe
tipofthehomogenizersubmerged,andbyholdingtheimmersedtiptoonesideofthetube.Rotorstatorhomogenizersare
availableindifferentsizesandoperatewithprobesofdifferentsizes.Probeswithdiametersof5mmand7mmaresuitable
forvolumesupto300landcanbeusedforhomogenizationinmicrofugetubes.Probeswithadiameterof10mmor
aboverequirelargertubes.
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Disruptionusingbeadmills
Indisruptionusingabeadmill,thesampleisagitatedathighspeedinthepresenceofbeads.Disruptionoccursbythe
shearingandcrushingactionofthebeadsastheycollidewiththecells.Disruptionefficiencyisinfluencedby:
Sizeandcompositionofbeads
Ratioofbuffertobeads
Amountofstartingmaterial
Speedandconfigurationofagitator
Disintegrationtime
Theoptimalbeadstouseare0.1mm(meandiameter)glassbeadsforbacteria,0.5mmglassbeadsforyeastand
unicellularanimalcells,37mmstainlesssteelbeadsforanimaltissues,and37mmstainlesssteelortungstencarbide
beadsforplantandfungaltissues.Itisessentialthatglassbeadsarepretreatedbywashinginconcentratednitricacid.
Alternatively,usecommerciallyavailableacidwashedglassbeads.Allotherdisruptionparametersmustbedetermined
empiricallyforeachapplication.
Disruptionusingamortarandpestle
Fordisruptionusingamortarandpestle,freezethesampleimmediatelyinliquidnitrogenandgrindtoafinepowderunder
liquidnitrogen.Transferthesuspension(tissuepowderandliquidnitrogen)intoaliquidnitrogencooled,appropriately
sizedtubeandallowtheliquidnitrogentoevaporatewithoutallowingthesampletothaw.Addlysisbufferandcontinueas
quicklyaspossiblewiththeisolationprocedure.
SpecialconsiderationsforisolatinggenomicDNAfromdifferentsamplesources
SomesamplesourcescontainsubstancesthatcancauseproblemsinDNAisolationandanalysis.Specialconsiderations
arerequiredwhenworkingwiththesesamplesources.Inthissection,considerationsforworkingwithanumberofdifferent
sourcesarediscussed.
Blood
Humanbloodsamplesareroutinelycollectedforclinicalanalysis.Bloodcontainsanumberofenzymeinhibitorsthatcan
interferewithdownstreamDNAanalysis.Inaddition,commonanticoagulantssuchasheparinandEDTAcaninterferewith
downstreamassays.DNAisolationfrombloodrequiresamethodtoprovidehighqualityDNAwithoutcontaminantsor
enzymeinhibitors.
Inanimals,erythrocytes(redbloodcells)frombirds,fish,andfrogscontainnucleiandhencegenomicDNA,whilethose
frommammalsdonot.Sincehealthymammalianbloodcontainsapproximately1000timesmoreerythrocytesthannuclei
containingleukocytes(whitebloodcells,comprisinglymphocytes,monocytes,andgranulocytes)removingtheerythrocytes
priortoDNAisolationcangivehigherDNAyields.Thiscanbeaccomplishedbyseveralmethods.Oneisselectivelysisof
erythrocytes,whicharemoresusceptiblethanleukocytestohypotonicshockandburstrapidlyinthepresenceofa
hypotonicbuffer.Alternatively,Ficolldensitygradientcentrifugationcanbeperformedtorecovermononuclearcells
(lymphocytesandmonocytes)andremoveerythrocytes.Thistechniquealsoremovesgranulocytes.Athirdmethodisto
preparealeukocyteenrichedfractionofwholeblood,calledbuffycoat,bycentrifugingwholebloodat3300xgfor10
minutesatroomtemperature.Aftercentrifugation,threedifferentfractionsaredistinguishable:theupperclearlayeris
plasmatheintermediatelayerisbuffycoatandthebottomlayercontainsconcentratederythrocytes.
Bloodsamples,includingthosetreatedtoremoveerythrocytes,canbeefficientlylysedusinglysisbufferandproteaseor
proteinaseK.AlongwiththeanimalsgenomicDNA,viralandbacterialDNAcanalsobeisolatedfrombloodsamples.
Otherclinicalsamples
MostbiologicalfluidscanbetreatedinthesamewayasbloodsamplesforisolationofDNA.IsolationofDNAfromstool
samplesismoredifficult,asstooltypicallycontainsmanycompoundsthatcandegradeDNAandinhibitdownstream
enzymaticreactions.
Animaltissuesandcellculture
AnimalcellculturesandmostanimaltissuescanbeefficientlylysedusinglysisbufferandproteaseorproteinaseK.Fresh
orfrozensamplesshouldbecutintosmallpiecestoaidlysis.Mechanicaldisruptionusingahomogenizerormortarand
pestlepriortolysiscanreducelysistime.Skeletalmuscle,heart,andskintissuehaveanabundanceofcontractileproteins,
connectivetissue,andcollagen,andcareshouldbetakentoensurecompletedigestionwithproteaseorproteinaseK.
Forfixedtissues,thefixativeshouldberemovedpriortolysis.Formalincanberemovedbywashingthetissuein
phosphatebufferedsaline(PBS).Paraffinshouldbesimilarlyremovedfromparaffinembeddedtissuesbyextractionwith
xylenefollowedbywashingwithethanol.
Yeastcellcultures
Yeastcellculturesmustfirstbetreatedwithlyticaseorzymolasetodigestthecellwall.Theresultingspheroplastsare
collectedbycentrifugationandthenlysedusinglysisbufferandproteinaseKorprotease.
BacterialDNA
ManybacterialcellculturescanbeefficientlylysedusinglysisbufferandproteaseorproteinaseK.Somebacteria,
particularlyGrampositivebacteria,requirepreincubationwithspecificenzymes(e.g.,lysozymeorlysostaphin)tolysethe
rigid,multilayeredcellwall.
BacterialDNAcanalsobeisolatedfromawidevarietyofclinicalsamples.Bacterialcellsshouldbepelletedfrombiological
fluids,andtheDNAisolatedasforbacterialcellcultures.Swabsamplesshouldbepretreatedwithfungicidebefore
centrifugationofbacterialcells.
DNAviruses
Inclinicalapplications,viralDNAisoften(althoughnotalways)isolatedfromcellfreebodyfluids,wheretheirtitercanbe
verylow.VirusparticlesmayneedtobeconcentratedbeforeDNAisolationbyultracentrifugation,ultrafiltration,or
precipitation.AdditionofcarrierDNAmayalsobenecessaryduringDNAisolationwhentheexpectedyieldofDNAislow.
IntegratedviralDNAispreparedusingthesameprocedureasforisolationofgenomicDNAfromtherelevantsample.
Bacteriophage,suchasM13andlambda,areisolatedfrominfectedbacterialcultures.Thebacterialcellsmustberemoved
fromtheculturebycentrifugationpriortoisolationofviralDNA.
Plants
IsolationofDNAfromplantmaterialpresentsspecialchallenges,andcommonlyusedtechniquesoftenrequireadaptation
beforetheycanbeusedwithplantsamples.Severalplantmetaboliteshavechemicalpropertiessimilartothoseofnucleic
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acids,andaredifficulttoremovefromDNApreparations.Copurifiedmetabolitesandcontaminantsintroducedbythe
purificationprocedure,suchassaltsorphenol,caninhibitenzymaticreactionsorcausevariationsinUV
spectrophotometricmeasurementsandgelmigration.
DNAisolationisoftenimprovedbyusingplantsgrownunderconditionsthatdonotinducehighlevelsofplantmetabolites.
Becauseofthegreatvariationamongplants,itisdifficulttomakegeneralstatementsaboutgrowthconditionstouse.
However,asageneralguideline,itisrecommendedtousehealthy,youngtissueswhenpossible.DNAyieldsfromyoung
tissuesareoftenhigherthanfromoldtissuebecauseyoungtissuegenerallycontainsmorecellsthanthesameamountof
oldertissue.Youngtissueofthesameweightalsocontainsfewermetabolites.Inaddition,manyprotocolsforhomemade
DNAisolationmethodsrecommendgrowingplantsindarknessfor12daysbeforeharvestingtopreventhighlevel
accumulationofplantmetabolites.
WorkingwithDNA:Goodmicrobiologicalpractice
Backtotop
GrowthofE.colistrains
GoodmicrobiologicaltechniquewillalwaysensurethebestyieldandqualityofplasmidDNA.Topreparetheperfect
bacterialcultureforyourplasmidprep,followthestepsbelow.
1.Prepareastarterculturebyinoculatingasinglecolonyfromafreshlystreakedselectiveplateinto210mlLB(Luria
Bertani)mediumcontainingtheappropriateantibiotic.Growat37Cfor~8hours(logarithmicgrowthphase,seefigure
GrowthofE.colicultures)withvigorousshaking(~300rpm).
Tip:Donotinoculatedirectlyfromglycerolstocks,agarstabs,orplatesthathavebeenstoredforalongtime,asthis
mayleadtolossormutationoftheplasmid.
Tip:Itisoftenconvenienttogrowthestartercultureduringthedaysothatthelargerculturecanbegrownovernightfor
harvestingthefollowingmorning.
2.Dilutethestarterculture1/500to1/1000intoalargervolumeofselectiveLBmedium,asindicatedintheappropriate
plasmidpurificationprotocol.
Useaflaskofatleast4timesthevolumeofculturetoensuresufficientaeration.
Donotusealargerculturevolumethanrecommendedintheprotocol,asthiswillresultininefficientlysisandreduce
thequalityofthepreparation.
3.Growthecultureat37Cwithvigorousshaking(~300rpm)for1216hours(seenextsection).
4.Harvestthebacterialculture1216hoursafterinoculation.Thiscorrespondstothetransitionfromlogarithmicinto
stationarygrowthphase(seefigure GrowthcurveofE.coliinLBmedium),whencelldensityishigh(34x109 cells
perml)andRNAcontentofcellsislow.HarvestingtooearlymayresultinlowerthanexpectedyieldsofplasmidDNA
duetoalowercelldensity.HarvestingtoolatemayresultinlowplasmidqualityandyieldduetoDNAdegradation
fromoveragingoftheculture.
Tip:Growthofculturesisdependentonfactorssuchashoststrain,plasmidinsertandcopynumber,andculture
medium.Todeterminetheoptimalharvestingtimeforaparticularsystem,monitorthecelldensityandthegrowthofthe
culturebymeasuringtheOD600 (seenextsection).
5.Harvestthebacterialculturebycentrifugationat6000xgfor15minat4C.Removealltracesofsupernatantby
invertingtheopencentrifugetubeuntilallofthemediumhasbeendrained.Thecellsarenowreadyforthelysis
procedure,asindicatedintheappropriateplasmidpurificationprotocol.
Theproceduremaybestoppedatthispointandcontinuedlaterbyfreezingthecellpelletsobtainedbycentrifugation.
Thefrozencellpelletsmaybestoredat20Cforseveralweeks.
TheE.coligrowthcurve
ThegrowthcurveofanE.coliculturecanbedividedintoseveraldistinctphases.Thefirst,lagphase,occursdirectlyafter
dilutionofthestartercultureintofreshmedium.Duringthisphase,celldivisionisslowasthebacteriaadapttothefresh
medium.Thebacteriathenstarttodividemorerapidlyandthecultureenterslogarithmic(log)phase(45hoursafter
dilution),duringwhichthenumberofcellsincreasesexponentially.Astheavailablenutrientsinthemediumareusedup
andreleasedmetabolitesinhibitbacterialgrowth,theculturebecomessaturatedandentersstationaryphase(~16hours
afterdilution),duringwhichcelldensityremainsconstant.Eventuallythecultureentersthephaseofdeclineascellsstartto
lyse,thenumberofviablebacteriafalls,andDNAbecomespartlydegraded.
StorageofE.colistrains
Backtotop
TherearedifferentmethodsforstoringE.colistrainsdependingonthedesiredstoragetime.Glycerolstocksandstab
culturesenablelongtermstorageofbacteria,whileagarplatescanbeusedforshorttermstorage.Preparationinstructions
andusefultipsforeachofthesemethodsaregivenbelow.
Glycerolstocks
E.colistrainscanbestoredformanyyearsat70Cin15%glycerol.
Prepareglycerolstocksofbacteriaasfollows:
1.Add0.15mlglycerol(100%)toa2mlscrewcapvialandsterilizebyautoclaving.
Tip:Vialsofsterilizedglycerolcanbepreparedinbatchesandstoredatroomtemperatureuntilrequired.
2.Add0.85mlofalogarithmicphaseE.coliculturetothevialofpresterilizedglycerol.
3.Vortexthevialvigorouslytoensureevenmixingofthebacterialcultureandtheglycerol.
4.Freezeinethanoldryiceorliquidnitrogenandstoreat70C.
Tip:Avoidrepeatedthawingandrefreezingofglycerolstocksasthiscanreducetheviabilityofthebacteria.
Tip:Forpreciousstrains,storageof2stockvialsisrecommended.
Tip:Whenrecoveringastoredstrain,itisadvisabletochecktheantibioticmarkersbystreakingthestrainontoa
selectiveplate.
Stabcultures
E.colistrainscanalsobestoredforupto1yearasstabsinsoftagar.Stabculturesareusedtotransportorsendbacterial
strainstootherlabs.
Preparestabculturesasfollows:
1.PrepareandautoclaveLBagar(standardLBmediumcontaining0.7%agar).
2.CooltheLBagartobelow50C(whenyoucanholditcomfortably)andaddtheappropriateantibiotic(s).Whilethe
agarisstillliquid,add1mlagartoa2mlscrewcapvialundersterileconditions,thenleavetosolidify.
3.Vialsofagarcanbepreparedinbatchesandstoredatroomtemperatureuntilrequired.
4.Usingasterilestraightwire,pickasinglecolonyfromafreshlystreakedplateandstabitdeepdownintothesoftagar
severaltimes(seefigure Inoculatingastabculture).
5.Incubatethevialat37Cfor812hleavingthecapslightlyloose.
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6.Sealthevialtightlyandstoreinthedark,preferablyat4C.
7.Whenrecoveringastoredstrain,itisadvisabletochecktheantibioticmarkersbystreakingthestrainontoaselective
plate.
Agarplates
PlatesofstreakedbacteriacanbesealedwithParafilmandstoredupsidedownat4Cforseveralweeks.Bacteriashould
alwaysbestreakedontoplatescontainingtheappropriateantibiotictoensurethatselectivemarkersarenotlost.
Toobtainwellisolatedcolonies,streakanagarplateasfollows:
1.Flameawireloop,andcoolonasparesterileagarplate.
2.Usingthewireloop,streakaninoculumofbacteria(fromaglycerolstock,stabculture,orsinglecolonyonanother
plate)acrossonecornerofafreshagarplate,asshowninthefigure Streakingbacteriaonagarplates.
3.Flameandcoolthewireloopagain.Passitthroughthefirststreakandthenstreakagainacrossafreshcornerofthe
plate.
4.Repeatagaintoformapattern.
5.Incubatetheplateupsidedownat37Cfor1224hoursuntilcoloniesdevelop.
Generatingliquidculturesfrombacterialstocks
Thefigure, EssentialstepsforstorageandhandlingofE.colishowsthesequenceofstepsnecessarytogofromastored
stockofbacteriatoaliquidcultureforplasmidisolation.Bacterialstocksshouldalwaysbestreakedontoselectiveplates
priortouse,tocheckthattheygiverisetohealthycoloniescarryingtheappropriateantibioticresistance.Stockscan
potentiallycontainmutantsarisingfromtheculturesusedtopreparethem,orcandeteriorateduringstorage.
Inoculateliquidculturesfromahealthy,wellisolatedcolony,pickedfromafreshlystreakedselectiveplate.Thiswillensure
thatcellsgrowingintheculturearealldescendedfromasinglefoundercell,andhavethesamegeneticmakeup.
Tip:Culturevolumes>10mlshouldnotbeinoculateddirectlyfromaplate,butdiluted1/500to1/1000fromaprecultureof
25ml.
Plasmidspecifications
Backtotop
Plasmidsvarywidelyintheircopynumber(seetable Originofreplicationandcopynumbersofvariousplasmidsand
cosmids),dependingontheoriginofreplicationtheycontain(pMB1orpSC101forexample)whichdetermineswhether
theyareunderrelaxedorstringentcontrolaswellasthesizeoftheplasmidanditsassociatedinsert.Someplasmids,
suchasthepUCseriesandderivatives,havemutationswhichallowthemtoreachveryhighcopynumberswithinthe
bacterialcell.PlasmidsbasedonpBR322andmanycosmidsaregenerallymaintainedatlowercopynumbers.Verylarge
plasmidsareoftenmaintainedatverylowcopynumberspercell.
Originofreplicationandcopynumberofvariousplasmidsandcosmids
DNAconstruct
Originofreplication
Copynumber
Classification
Plasmids
pUCvectors
pMB1*
500700
Highcopy
pBluescriptvectors
pGEMvectors
ColE1
pMB1*
300500
300400
Highcopy
Highcopy
pTZvectors
pBR322andderivatives
pMB1*
pMB1*
>1000
1520
Highcopy
Lowcopy
pQEvectors
ColE1
~30
Lowcopy
pREP4
pACYCandderivatives
P15A
P15A
~30
1012
Lowcopy
Lowcopy
pSC101andderivatives
Cosmids
pSC101
~5
Verylowcopy
SuperCos
pMB1*
1020
Lowcopy
pWE15
ColE1
1020
Lowcopy
*ThepMB1originofreplicationiscloselyrelatedtothatofColE1andfallsinthesameincompatibilitygroup.Thehighcopyplasmidslisted
herecontainmutatedversionsofthisorigin.
Bacterialcultivationmediaandantibiotics
Backtotop
Liquidmedia
LiquidculturesofE.colicangenerallybegrowninLB(LuriaBertani)medium.Pleasenote,however,thatanumberof
differentLBbroths,withdifferentcompositions,arecommonlyused.Differentformulationscontaindifferentconcentrations
ofNaClandgiverisetovariedyieldsofplasmidDNA.WerecommendusingtheLBcompositioninthetable LBmediato
obtainhighestyieldsofplasmidDNA.
LBmedia
Component
Amountperliter
Tryptone
10g
Yeastextract
NaCl
5g
10g
Forpreparationof1literofLBmedium,add10gNaCl,10gtryptone,and5gyeastextractto950mldistilledordeionized
water,andshakeorstiruntildissolved.AdjustthepHto7.0with5MNaOH.Adjustthevolumeofthesolutionto1literwith
distilledordeionizedwater.Decantintosmalleraliquotsandsterilizebyautoclaving(see Sterilizingmedia).
Tip:Itisadvisabletoautoclaveliquidmediuminseveralsmallbottlesratherthaninonelargevesseltoavoidpossible
contaminationofanentirebatch.Afterautoclaving,donotusemediumfor24hourstoensurethatitisproperlysterilized
andfreeofcontaminatingmicroorganisms.
Tip:Antibioticsshouldbeaddedtoliquidmediumimmediatelypriortousefromstockantibioticsolutionsthathavebeen
filtersterilized,distributedintoaliquots,andstoredinthedarkat20C(see Antibiotics).
Sterilizingmedia
Sterilizeliquidorsolidmediabyautoclaving,usingapressureandtimeperiodsuitableforthetypeofmedium,bottlesize,
andautoclavetype.
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Tip:Fillbottlesonly3/4fullwithmediumandloosenthecapsbeforeautoclavingtoavoidhotmediumboilingover.Tighten
capsoncethemediaiscool(<40C)tokeepitcompletelysterile.
Tip:Antibioticsandnutrientssuchasaminoacidsareinactivatedbythehightemperaturesofanautoclave.Theyshouldbe
sterilizedbyfiltrationthroughafilterunitwithaporesizeof0.2m,andaddedtothecooled,autoclavedmediumfrom
properlystoredstocksolutions.
Solidmedia
E.colistrainscangenerallybestreakedandstoredonLBplatescontaining1.5%agarandtheappropriateantibiotic(s).
Preparation:PrepareLBmediumaccordingtothecompositiongivenin Liquidmedia.Justbeforeautoclaving,add15g
agarperliterandmix.Afterautoclaving,swirlthemediumgentlytodistributethemeltedagarevenlythroughoutthe
solution.Takecarethatthehotliquiddoesnotboiloverwhenswirled.
Tip:Coolautoclavedagarmediumtobelow50C(whenyoucanholditcomfortably)beforeaddingheatsensitive
antibioticsandnutrients.Mixthoroughlytoobtainanevenconcentrationthroughoutthemediumbeforepouring.
Tip:Pourplatesinalaminarflowhoodor,ifnohoodisavailable,onacleanedbenchsurfacenexttoaBunsen.Use3035
mlmediumperstandard90mmpetridish(~30platesperliterofmedium).
Afterpouringplates,anyairbubblesmayberemovedbypassingtheflameofaBunsenburnerbrieflyoverthesurface.Do
notlingerwiththeflameasthismaydestroyantibioticsinsectionsoftheplates.
Dryplateseitherdirectlyaftersolidificationorjustbeforeusebyremovingthelidsandstandingtheplatesinalaminarflow
hoodfor1hour.Alternatively,ifyoudonothaveaccesstoahood,platescanbedriedwiththecoversslightlyopenina
37Cincubatorfor30min,orleftupsidedownwithlidsonatroomtemperaturefor23days.
Tip:Storeplatesinvertedat4Cinadarkroomorwrappedinaluminumfoiltopreservelightsensitiveantibiotics.Donot
storeforlongerthan3monthsasantibioticsmaydegrade.
Antibiotics
Bacterialstrainscarryingplasmidsorgeneswithantibioticselectionmarkersshouldalwaysbeculturedinliquidoronsolid
mediumcontainingtheselectiveagent.Lackofantibioticselectioncanleadtolossoftheplasmidcarryingthegenetic
markerandpotentiallytoselectionoffastergrowingmutants!
Tip:Preparestocksolutionsofantibioticsseparatelyfrombatchesofliquidorsolidmedia,sterilizebyfiltration,aliquot,and
storeinthedarkat20C.Recommendedstockandworkingconcentrationsforcommonlyusedantibioticsareshownin
thetable Concentrationsofcommonlyusedantibiotics.
Tip:Beforeaddingantibioticstofreshlyautoclavedmedium,ensurethatthemediumhascooledtobelow50C.
Concentrationsofcommonlyusedantibiotics
Antibiotic
Stocksolutionconcentration
Storagetemperature
Workingconcentration(dilution)
Ampicillin(sodiumsalt)
Chloramphenicol
50mg/mlinwater
30mg/mlinethanol
20C
20C
100g/ml(1/500)
170g/ml(1/200)
Kanamycin
Streptomycin
10mg/mlinwater
50mg/mlinwater
20C
20C
50g/ml(1/200)
50g/ml(1/200)
TetracyclineHCl
5mg/mlinethanol
20C
50g/ml(1/100)
Lysisofbacterialcellsforplasmidpurification
Backtotop
EffectivelysisofbacterialcellsisakeystepinplasmidisolationasDNAyieldandqualitydependonthequalityofcell
lysateusedforthepurification.
Alkalinelysis
Alkalinelysisisoneofthemostcommonlyusedmethodsforlysingbacterialcellspriortoplasmidpurification(4,5).
Productionofalkalinelysatesinvolvesfourbasicsteps(seefigure Theprincipleofalkalinelysis).
1.ResuspendharvestedbacterialcellsinTrisClEDTAbuffercontainingRNaseA.
Tip:Ensurethatbacteriaareresuspendedcompletelyleavingnocellclumpsinordertomaximizethenumberofcells
exposedtothelysisreagents.
Tip:Forlargescalepurificationoflowcopyplasmids,forwhichlargerculturesvolumesareused,itmaybebeneficial
toincreasethelysisbuffervolumesinordertoincreasetheefficiencyofalkalinelysisandtherebytheDNAyield.
2.LysecellsusingNaOH/SDS.Sodiumdodecylsulfate(SDS)solubilizesthephospholipidandproteincomponentsof
thecellmembrane,leadingtolysisandreleaseofthecellcontents.NaOHdenaturesthechromosomalandplasmid
DNA,aswellasproteins.ThepresenceofRNaseAensuresthatliberatedcellularRNAisdigestedduringlysis.
Tip:Ifafteradditionoflysisbuffer(NaOH/SDS)thesolutionappearsveryviscousandisdifficulttomix,thisindicates
excessbiomassinthelysatestep.Thisresultsininsufficientcelllysisanditisrecommendedtodoubletheamountof
lysisandneutralizationbuffersused.
Tip:Avoidvigorousstirringorvortexingofthelysateasthiscanshearthebacterialchromosome,whichwillthen
copurifywiththeplasmidDNA.Thesolutionshouldbemixedgentlybutthoroughlybyinvertingthelysisvessel46
times.
Tip:Donotallowthelysistoproceedforlongerthan5minutes.ThisisoptimalforreleaseoftheplasmidDNA,while
avoidingirreversibleplasmiddenaturation.
3.Neutralizethelysatebyaddingacidicpotassiumacetate.Note:Thehighsaltconcentrationcausespotassiumdodecyl
sulfate(KDS)toprecipitate,anddenaturedproteins,chromosomalDNA,andcellulardebrisarecoprecipitatedin
insolublesaltdetergentcomplexes.PlasmidDNA,beingcircularandcovalentlyclosed,renaturescorrectlyand
remainsinsolution.
Tip:Precipitationcanbeenhancedbyusingchilledneutralizationbufferandincubatingonice.
4.Clearthelysatebyeithercentrifugationorfiltration,toprecipitatethedebris.
Note:PurificationofplasmidDNAfromclearedbacteriallysateswastraditionallyperformedusingcesiumchloride
(CsCl)ultracentrifugation.Today,avarietyofcommerciallyavailableplasmidpurificationkitsoffereasyproceduresfor
differentthroughputrequirementsandapplications.
Otherlysismethods
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Anumberofothermethodshavebeendescribedforlysingbacterialcells(1,6).Someofthesemethodsweredeveloped
forotherapplicationsandmaynotbesuitableforplasmidDNApreparation.
Boilinglysis:Bacterialcellsaretreatedwithlysosometoweakenthecellwallsandthenlysedbyheatinginaboiling
waterbathfor~1minute.
Lysiswithdetergent:Bacterialcellsarelysedbytreatmentwithandionicdetergent(e.g.,SDS)oranonionic
detergent(e.g.,TritonX100).
Mechanicallysis:Bacterialcellsarelysedbymechanicaldisruption(e.g.,bysonification).
Enzymaticdigestion:Somelysismethodsincludetreatmentofbacteriawithenzymessuchaslysozymewhichassist
inweakeningcellwalls.
LysisofbacteriaotherthanE.coli
IsolationofplasmidDNAfrombacteriaotherthanE.coliusuallyrequiresmodificationstothelysisprocedureinorderto
optimizelysisconditionsfortheparticularspecies.
TransformationofDNA
Backtotop
PreparationofcompetentE.coli
CellsthathavetheabilitytotakeupDNA(fromavarietyofsources)aretermedcompetent.Severaltechniquesexistto
preparecompetentcellsandonesuchtechniqueforpreparingcompetentE.coliisgivenbelow.
Note:Cellspreparedusingthisprotocolarenotsuitableforelectroporation.
Materialsrequired
E.colicellsinglycerolstockvial
LBmedium
LBagarplates
Appropriateselectiveantibiotics
TFB1buffer(seetable, BufferTFB1)
TFB2buffer(seetable, BufferTFB2)
BufferTFB1
Workingsolution,pH5.8
Component
Amountperliter
100mMRbCl
50mMMnCl2
RbCl
MnCl24H2 O
12.1g
30mMpotassiumacetate
Potassiumacetate
2.9g
10mMCaCl2
CaCl2
1.1g
15%glycerol
Glycerol
15ml
9.9g
AdjustpHto5.5andsterilizebyfiltration.
BufferTFB2
Workingsolution,pH6.8
Component
Amountperliter
100mMMOPS
50mMRbCl
MOPS
RbCl
2.1g
1.2g
75mMCaCl2
CaCl2
8.3g
15%glycerol
Glycerol
15ml
AdjustpHto6.5withKOHandsterilizebyfiltration.
1.RemoveatraceofE.colicellsfromtheglycerolstockvialwithasteriletoothpickorinoculatingloop,andstreakitout
onLBagarplatescontaininganappropriateconcentrationoftherelevantselectiveantibiotic(s)(see Antibiotics).If
thehoststrainhasalreadybeenculturedandstoredat28C(culturescanbestoredat28Cforupto3months
withoutanysignificantlossofviability),streakoutbacteriafromthosestocks.
2.Incubateat37Covernight.
3.Pickasinglecolonyandinoculate10mlLBmediumcontainingrelevantantibiotic(s).Growovernightat37C.
4.Add1mlovernightcultureto100mlprewarmedLBmediumcontainingtherelevantantibiotic(s)ina500mlflask,and
shakeat37CuntilanOD600 of0.5isreached(approximately90120min).
5.Coolthecultureonicefor5min,andtransfertheculturetoasterile,roundbottomcentrifugetube.
6.Collectthecellsbycentrifugationatlowspeed(5min,4000xg,4C).
7.Discardthesupernatantcarefully.Alwayskeepthecellsonice.
8.Resuspendthecellsgentlyincold(4C)TFB1buffer(30mlfora100mlculture)andkeepthesuspensiononicefor
anadditional90min.
9.Collectthecellsbycentrifugation(5min,4000xg,4C).
10.Discardthesupernatantcarefully.Alwayskeepthecellsonice.
11.Resuspendthecellscarefullyin4mlicecoldTFB2buffer.
12.Preparealiquotsof100200linsterilemicrocentrifugetubesandfreezeinliquidnitrogenoradryiceethanolmix.
Storethecompetentcellsat70C.
TransformationofcompetentE.coli
Backtotop
TransformationistheprocessinwhichplasmidDNAisintroducedintoabacterialhostcell.Severalmethodsexistfor
transformationofbacterialcells,oneofwhichisgivenbelow.
CompetentE.colicells(see PreparationofcompetentE.coli)
SOCmedium(seetable SOCmedium)
LBagarplates(see Solidmedia)
SOCmedium
Component
Amountperliter
Tryptone
Yeastextract
20g
5g
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NaCl
0.5g
Dissolve,thenadd:
250mMKCl
10ml
2MMgCl2
5ml
Autoclave,cool,thenadd:
1Msterileglucose
20ml
Donotsterilizebyautoclavingfilterthesolutionthrougha0.2mfilterinstead.
1.TransferanaliquotoftheDNAtobetransformed(10lorless)intoacoldsterile1.5mlmicrocentrifugetube,andkeep
itonice.
2.ThawanaliquotoffrozencompetentE.colicellsonice.
3.Gentlyresuspendthecellsandtransfer100lofthecellsuspensionintothemicrocentrifugetubewiththeplasmid
DNA,mixcarefully,andkeeponicefor20min.
4.Transferthetubetoa42Cwaterbathorheatingblockfor90s.
5.Add500lSOCmediumtothecellsandincubatefor6090minat37C.
Tip:Shakingincreasestransformationefficiency.
6.Plateout50,100,and200laliquotsonLBagarplatescontainingtherelevantantibiotic(s).Incubatetheplatesat
37Covernightuntilcoloniesdevelop.
Positivecontroltochecktransformationefficiency
Transformcompetentcellswith1ngofacontrolplasmidcontaininganantibioticresistancegene.PlateontoLBagar
platescontainingtherelevantantibiotic(s).Comparethenumberofcoloniesobtainedwiththecontrolplasmidtothe
numberobtainedwiththeplasmidofinteresttocomparetransformationefficiency.
Negativecontroltocheckantibioticactivity
Transformcellswith20lofTE.Plateatleast200lofthetransformationmixonasingleLBagarplatecontainingthe
relevantantibiotic(s).Anabsenceofcoloniesontheplatesindicatesthattheantibioticisactive.
IsopropanolprecipitationofDNA
Backtotop
Alcoholprecipitationiscommonlyusedforconcentrating,desalting,andrecoveringnucleicacids.Precipitationismediated
byhighconcentrationsofsaltandtheadditionofeitherisopropanolorethanol.Sincelessalcoholisrequiredfor
isopropanolprecipitation,thisisthepreferredmethodforprecipitatingDNAfromlargevolumes.Inaddition,isopropanol
precipitationcanbeperformedatroomtemperature,whichminimizescoprecipitationofsaltthatinterfereswith
downstreamapplications.
1.Adjustthesaltconcentrationifnecessary,forexample,withsodiumacetate(0.3M,pH5.2,finalconcentration)or
ammoniumacetate(2.02.5M,finalconcentration).
2.Add0.60.7volumesofroomtemperatureisopropanoltotheDNAsolutionandmixwell.
Tip:Useallsolutionsatroomtemperaturetominimizecoprecipitationofsalt.
Tip:Donotusepolycarbonatetubesforprecipitationaspolycarbonateisnotresistanttoisopropanol.
3.Centrifugethesampleimmediatelyat10,00015,000xgfor1530minat4C.
Tip:Centrifugationshouldbecarriedoutat4Ctopreventoverheatingofthesample.(Whenprecipitatingfromsmall
volumes,centrifugationmaybecarriedoutatroomtemperature.)
Tip:GenomicDNAcanalternativelybeprecipitatedbyspoolingtheDNAusingaglassrodfollowingadditionof
isopropanol.ThespooledDNAshouldbetransferredimmediatelytoamicrofugetubecontaininganappropriatebuffer
andredissolved(seestep9).
4.Carefullydecantthesupernatantwithoutdisturbingthepellet.
Tip:Markingtheoutsideofthetubebeforecentrifugationallowsthepellettobemoreeasilylocated.Pelletsfrom
isopropanolprecipitationhaveaglassyappearanceandmaybemoredifficulttoseethanthefluffysaltcontaining
pelletsresultingfromethanolprecipitation.
Tip:Careshouldbetakenwhenremovingthesupernatantaspelletsfromisopropanolprecipitationaremoreloosely
attachedtothesideofthetube.
Tip:Carefullytipthetubewiththepelletontheuppersidetoavoiddislodgingthepellet.
Tip:Forvaluablesamples,thesupernatantcanberetaineduntilrecoveryoftheprecipitatedDNAhasbeenverified.
5.WashtheDNApelletbyadding110ml(dependingonthesizeofthepreparation)ofroomtemperature70%ethanol.
Thisremovescoprecipitatedsaltandreplacestheisopropanolwiththemorevolatileethanol,makingtheDNAeasier
toredissolve.
6.Centrifugeat10,00015,000xgfor515minat4C.
Tip:CentrifugethetubeinthesameorientationaspreviouslytorecovertheDNAintoacompactpellet.
7.Carefullydecantthesupernatantwithoutdisturbingthepellet.
8.Airdrythepelletfor520min(dependingonthesizeofthepellet).
Tip:Donotoverdrythepellet(e.g.,byusingavacuumevaporator)asthiswillmakeDNA,especiallyhighmolecular
weightDNA,difficulttoredissolve.
9.RedissolvetheDNAinasuitablebuffer.
Tip:ChooseanappropriatevolumeofbufferaccordingtotheexpectedDNAyieldandthedesiredfinalDNA
concentration.
Tip:UseabufferwithapHof7.58.0,asDNAdoesnotdissolveeasilyinacidicbuffers.(Ifusingwater,checkpH.)
Tip:RedissolvebyrinsingthewallstorecoveralltheDNA,especiallyifglasstubeshavebeenused.Toavoid
shearingtheDNA,donotpipetorvortex.
Tip:HighmolecularweightDNA,suchasgenomicDNA,shouldberedissolvedverygentlytoavoidshearing,e.g.,at
roomtemperatureovernightorat55Cfor12hwithgentleagitation.
StorageofDNA
Backtotop
PurifiedDNAshouldbestoredat20Cor70Cunderslightlybasicconditions(e.g.,TrisCl,pH8.0orTEbuffersee
tables 1MTrisCland TEbuffer)becauseacidicconditionscancausehydrolysisofDNA.Avoidrepeatedfreeze
thawingasthiswillleadtoprecipitates.
Dilutedsolutionsofnucleicacids(e.g.,dilutionseriesusedasstandards)shouldbestoredinaliquots(insiliconizedtubes,
wherepossible)andthawedonceonly.Thisavoidsadsorptionofnucleicacidstothetubewalls,whichwouldreducethe
concentrationofnucleicacidsinsolution.
1MTrisCl
Component
Amountperliter
Trisbase
121.1g
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AdjusttopHwithHCl.
TEbuffer
Component
Amountperliter
1MTrisCl,pH7.4
10ml
0.5MEDTA,pH8.0
2ml
Endotoxinsandwhattoconsider
Backtotop
Whatareendotoxins?
Endotoxins,alsoknownaslipopolysaccharidesorLPS,arecellmembranecomponentsofGramnegativebacteria(e.g.,E.
coli).Thelipidportionoftheouterlayeroftheoutermembraneiscompletelycomposedofendotoxinmolecules(seefigure
Bacterialcellwall).AsingleE.colicellcontainsabout2millionLPSmolecules,eachconsistingofahydrophobiclipidA
moiety,acomplexarrayofsugarresiduesandnegativelychargedphosphategroups(seefigure Schematicdiagramof
theendotoxinmolecule).Therefore,eachendotoxinmoleculepossesseshydrophobic,hydrophilic,andchargedregions
givingituniquefeatureswithrespecttopossibleinteractionswithothermolecules.Bacteriashedsmallamountsof
endotoxinsintotheirsurroundingswhiletheyareactivelygrowingandlargeamountswhentheydie.Duringlysisof
bacterialcellsforplasmidpreparations,endotoxinmoleculesarereleasedfromtheoutermembraneintothelysate.
Endotoxinssignificantlyreducetransfectionefficienciesinendotoxinsensitivecelllines.Furthermore,endotoxinscan
influencetheuptakeofplasmidDNAintransfectionexperimentsbycompetingwithDNAforfreetransfectionreagent.
Overall,endotoxinsrepresentanoncontrollablevariableintransfectionexperimentsetup.Theyareinvisibleonagarose
gelsandimpossibletodetectbyopticaldensityandinfluencetheoutcomeandreproducibilityofresultsandmakingthem
difficulttocompareandinterpret.
Endotoxincontaminationofdifferentplasmidpreparationmethods
Thechemicalstructureandpropertiesofendotoxinmoleculesandtheirtendencytoformmicellarstructuresleadto
copurificationofendotoxinswithplasmidDNA.Forexample,inCsClultracentrifugation,theCsClbandedDNAiseasily
contaminatedwithendotoxinmolecules,whichhaveasimilardensityinCsCltoplasmidethidiumbromidecomplexes.
Onsizeexclusionresins,thelargesizeofthemicellarformofendotoxincausesthemoleculetobehavelikealargeDNA
moleculeandinanionexchangechromatography,thenegativechargespresentontheendotoxinmoleculecaninteract
withanionexchangeresins,thusleadingtocopurificationofendotoxinswiththeplasmidDNA.
However,thelevelofendotoxincontaminationfoundinplasmidDNAisdependentonthepurificationmethodused.
Howareendotoxinsmeasured?
Historically,endotoxinsweremeasuredinaclottingreactionbetweentheendotoxinandaclottableproteininthe
amoebocytesofLimuluspolyphemus,thehorseshoecrab.
Todaymuchmoresensitivephotometrictests(e.g.,KineticQCLTestfromBioWhittaker,Inc.)areused,whicharebasedon
aLimulusamoebocytelysate(LAL)andasyntheticcolorproducingsubstrate.LPScontaminationisusuallyexpressedin
endotoxinunits(EU).Typically,1ngLPScorrespondsto110EU.
Influenceofendotoxinsonbiologicalapplications
EndotoxinsstronglyinfluencetransfectionofDNAintoprimarycellsandsensitiveculturedcells,andincreasedendotoxin
levelsleadtosharplyreducedtransfectionefficiencies.Furthermore,itisextremelyimportanttouseendotoxinfreeplasmid
DNAforgenetherapyapplications,sinceendotoxinscausefever,endotoxicshocksyndrome,andactivationofthe
complementcascadeinanimalsandhumans.
EndotoxinsalsointerferewithinvitrotransfectionintoimmunecellssuchasmacrophagesandBcellsbycausing
nonspecificactivationofimmuneresponses.Theseresponsesincludetheinducedsynthesisofimmunemediatorssuchas
IL1andprostaglandin.Itisimportanttomakesurethatplasticware,media,sera,andplasmidDNAarefreeofLPS
contaminationtoavoidmisinterpretationofexperimentalresults.
Endotoxinfreeplasticwareandglassware
ToavoidrecontaminationofplasmidDNAafterinitialendotoxinremoval,werecommendusingonlynewplasticwarewhich
iscertifiedtobepyrogenorendotoxinfree.Endotoxinfreeorpyrogenfreeplasticwarecanbeobtainedfrommany
differentsuppliers.
Endotoxinsadherestronglytoglasswareandaredifficulttoremovecompletelyduringwashing.Standardlaboratory
autoclavingprocedureshavelittleornoeffectonendotoxinlevels.Moreover,iftheautoclavehaspreviouslybeenusedfor
bacteria,theglasswarewillbecomeextensivelycontaminatedwithendotoxinmolecules.Heatingglasswareat180C
overnightisrecommendedtodestroyanyattachedendotoxinmolecules.
ItisalsoimportantnottorecontaminatethepurifiedendotoxinfreeDNAbyusingreagentsthatarenotendotoxinfree.
QuantificationofDNA
Backtotop
ReliablemeasurementofDNAconcentrationisimportantformanyapplicationsinmolecularbiology.Spectrophotometry
andfluorometryarecommonlyusedtomeasurebothgenomicandplasmidDNAconcentration.Spectrophotometrycanbe
usedtomeasuremicrogramquantitiesofpureDNAsamples(i.e.,DNAthatisnotcontaminatedbyproteins,phenol,
agarose,orRNA).Fluorometryismoresensitive,allowingmeasurementofnanogramquantitiesofDNA,andfurthermore,
theuseofHoechst33258dyeallowsspecificanalysisofDNA.
Spectrophotometry
DNAconcentrationcanbedeterminedbymeasuringtheabsorbanceat260nm(A260 )inaspectrophotometerusinga
quartzcuvette.Forgreatestaccuracy,readingsshouldbebetween0.1and1.0.Anabsorbanceof1unitat260nm
correspondsto50ggenomicDNAperml(A260 =1for50g/mlbasedonastandard1cmpathlength.Thisrelationis
validonlyformeasurementsmadeatneutralpH,therefore,samplesshouldbedilutedinalowsaltbufferwithneutralpH
(e.g.,TrisCl,pH7.0).Anexampleofthecalculationinvolvedinnucleicacidquantificationwhenusingaspectrophotometer
(see SpectrophotometricmeasurementofDNAconcentration).
WhenworkingwithsmallamountsofDNA,suchaspurifiedPCRproductsorDNAfragmentsextractedfromagarosegels,
quantificationviaagarosegelanalysismaybemoreeffective(see Agarosegel).
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Tip:Ifyouusemorethanonecuvettetomeasuremultiplesamples,thecuvettesmustbematched.
Tip:SpectrophotometricmeasurementsdonotdifferentiatebetweenDNAandRNA,soRNAcontaminationcanleadto
overestimationofDNAconcentration.
Tip:Phenolhasanabsorbancemaximumof270275nm,whichisclosetothatofDNA.Phenolcontaminationmimicsboth
higheryieldsandhigherpurity,becauseofanupwardshiftintheA260 value.
Effectsofsolventsonspectrophotometricreadings
Absorptionofnucleicacidsdependsonthesolventusedtodissolvethenucleicacid(7).A260 valuesarereproducible
whenusinglowsaltbuffer,butnotwhenusingwater.ThisismostlikelyduetodifferencesinthepHofthewatercausedby
thesolvationofCO2 fromair.A260 /A280 ratiosmeasuredinwateralsogiverisetoahighvariabilitybetweenreadings(see
figure EffectofsolventonA260 /A280 ratio)andtheratiosobtainedaretypically<1.8,resultinginreducedsensitivityto
proteincontamination(7).Incontrast,A260 /A280 ratiosmeasuredinalowsaltbufferwithslightlyalkalinepHaregenerally
reproducible.
EffectofRNAcontaminationonspectrophotometricreadings
DependingontheDNAisolationmethodused,RNAwillbecopurifiedwithgenomicDNA.RNAmayinhibitsome
downstreamapplications,butitwillnotinhibitPCR.SpectrophotometricmeasurementsdonotdifferentiatebetweenDNA
andRNA,soRNAcontaminationcanleadtooverestimationofDNAconcentration.RNAcontaminationcansometimesbe
detectedbyagarosegelanalysiswithroutineethidiumbromidestaining,althoughnotquantifiedeffectively.RNAbands
appearfaintandsmearyandareonlydetectedinamounts2530ng(0.5:1RNA:DNAratio).
TreatmentwithRNaseAwillremovecontaminatingRNAthiscaneitherbeincorporatedintothepurificationprocedureor
performedaftertheDNAhasbeenpurified.Priortouse,ensurethattheRNaseAsolutionhasbeenheattreatedtodestroy
anycontaminatingDNaseactivity.Alternatively,useDNasefreeRNasepurchasedfromareliablesupplier.
RNAcontaminationofplasmidDNAcanbeaconcerndependingonthemethodusedforplasmidpreparation.Methods
usingalkalinelysiswithphenolextractioncannotseparateRNAfromplasmidDNA,leadingtohighlevelsofRNA
contamination.AdvancedanionexchangetechnologyallowsisolationofhighmolecularweightgenomicDNAthatisfree
ofRNA.
PurityofDNA
Theratioofthereadingsat260nmand280nm(A260 /A280 )providesanestimateofDNApuritywithrespectto
contaminantsthatabsorbUVlight,suchasprotein.TheA260 /A280ratioisinfluencedconsiderablybypH.Sincewaterisnot
buffered,thepHandtheresultingA260 /A280ratiocanvarygreatly.LowerpHresultsinalowerA260 /A280 ratioandreduced
sensitivitytoproteincontamination(7).ForaccurateA260 /A280values,werecommendmeasuringabsorbanceinaslightly
alkalinebuffer(e.g.,10mMTrisCl,pH7.5).Besuretozerothespectrophotometerwiththeappropriatebuffer.
PureDNAhasanA260 /A280 ratioof1.71.9.Scanningtheabsorbancefrom220320nmwillshowwhetherthereare
contaminantsaffectingabsorbanceat260nm.Absorbancescansshouldshowapeakat260nmandanoverallsmooth
shape.
Fluorometry
FluorometryallowsspecificandsensitivemeasurementofDNAconcentrationbyuseofafluorescentdyewithcommon
dyesincludingHoechstdyesandPicoGreen.
Hoechst33258haslittleaffinityforRNA,allowingaccuratequantificationofDNAsamplesthatarecontaminatedwithRNA.
Itshowsincreasedemissionat458nmwhenboundtoDNA.DNAstandardsandsamplesaremixedwithHoechst33258
andmeasuredinglassoracryliccuvettesusingascanningfluorescencespectrophotometeroradedicatedfilter
fluorometersetatanexcitationwavelengthof365nmandanemissionwavelengthof460nm.Thesamplemeasurements
arethencomparedtothestandardstodetermineDNAconcentration.
Tip:AsHoechst33258preferentiallybindsATrichDNA,usestandardswithasimilarbasecompositiontothesampleDNA.
PicoGreenisahighlysensitivemeasureofdsDNAandcanmeasureaslittleas20pgdsDNAina200lassayvolume.
Indeed,DNAconcentrationsfrom500pg/mlto500ng/mlcanallbemeasuresusingasingledyeconcentration.Theassay
isoptimizedtominimizethefluorescencecontributionsofRNAandssDNA,suchthatdsDNAcanbeaccuratelyquantified
inthepresenceofequimolarconcentrationsofssDNAandRNAwithminimaleffectonthequantitativeresults.
Agarosegel
AgarosegelanalysisenablesquickandeasyquantificationofDNA,especiallyforsmallDNAfragments(suchasPCR
products).Aslittleas20ngDNAcanbedetectedbyagarosegelelectrophoresiswithethidiumbromidestaining.TheDNA
sampleisrunonanagarosegelalongsideknownamountsofDNAofthesameorasimilarsize.Theamountofsample
DNAloadedcanbeestimatedbycomparisonofthebandintensitywiththestandardseithervisually(seefigure Agarose
gelanalysisofplasmidDNA)orusingascannerorimagingsystem.Besuretousestandardsofroughlythesamesizeas
thefragmentofinteresttoensurereliableestimationoftheDNAquantity,sincelargefragmentsinterchelatemoredyethan
smallfragmentsandgiveagreaterbandintensity.
Morepreciseagarosegelquantificationcanbeachievedbydensitometricmeasurementofbandintensityandcomparison
withastandardcurvegeneratedusingDNAofaknownconcentration.Inmostexperimentstheeffectiverangefor
comparativedensitometricquantificationisbetween20and100ng.
Tip:TheamountofDNAusedfordensitometricquantificationshouldfallwithinthelinearrangeofthestandardcurve.
See DNAanalysisusinganalyticalgels,forfurtherinformationonagarosegelelectrophoresis.
RestrictionendonucleasedigestionofDNA
Backtotop
Principleofrestrictiondigestion
ManyapplicationsrequireconversionofgenomicDNAintoconvenientlysizedfragmentsbyrestrictionendonuclease
digestion.ThisyieldsDNAfragmentsofaconvenientsizefordownstreammanipulations.Restrictionendonucleasesare
bacterialenzymesthatbindandcleaveDNAatspecifictargetsequences.TypeIIrestrictionenzymesarethemostwidely
usedinmolecularbiologyapplications.TheybindDNAataspecificrecognitionsite,consistingofashortpalindromic
sequence,andcleavewithinthissite,e.g.,AGCT(forAluI),GAATTC(forEcoRI),andsoon.Isoschizomersaredifferent
enzymesthatsharethesamespecificity,andinsomecases,thesamecleavagepattern.
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Tip:Isoschizomersmayhaveslightlydifferentpropertiesthatcanbeveryuseful.Forexample,theenzymesMboIand
Sau3Ahavethesamesequencespecificities,butMboIdoesnotcleavemethylatedDNA,whileSau3Adoes.Sau3Acan
thereforebeusedinsteadofMboIwherenecessary.
Selectingsuitablerestrictionendonucleases
Thefollowingfactorsneedtobeconsideredwhenchoosingsuitablerestrictionenzymes:
Fragmentsize
Methylationsensitivity
Bluntended/stickyendedfragments
Compatibilityofreactionconditions(wheremorethanoneenzymeisused)
Fragmentsize
Restrictionenzymeswithshorterrecognitionsequencescutmorefrequentlythanthosewithlongerrecognitionsequences.
Forexample,a4basepair(bp)cutterwillcleave,onaverage,every44 (256)bases,whilea6bpcuttercleavesevery46
(4096)bases.
Tip:Use6bpcuttersformappinggenomicDNAorYACs,BACs,orP1s,asthesegivefragmentsinasuitablesizerangefor
cloning.
Methylation
ManyorganismshaveenzymescalledmethylasesthatmethylateDNAatspecificsequences.Notallrestrictionenzymes
cancleavetheirrecognitionsitewhenitismethylated.Thereforethechoiceofrestrictionenzymeisaffectedbyits
sensitivitytomethylation.Inaddition,methylationpatternsdifferindifferentspecies,alsoaffectingthechoiceofrestriction
enzyme.
TheCpGdinucleotideoccursabout5timeslessfrequentlyinmammalianDNAthanwouldbeexpectedbychance,
andmostrestrictionenzymeswithaCpGdinucleotideintheirrecognitionsitedonotcleaveifthecytosineis
methylated.ThereforemanyenzymeswithCpGintheirrecognitionsite,suchasEagI,NotI,andSalI,cleave
mammalianDNAonlyrarely.
Drosophila,Caenorhabditis,andsomeotherspeciesdonotpossessmethylatedDNA,andhaveahigherproportionof
CpGdinucleotidesthanmammalianspecies.Rarecutterenzymesthereforecleavemorefrequentlyinthesespecies.
PlantDNAishighlymethylated,soforsuccessfulmappinginplants,chooseenzymesthateitherdonotcontainaCpG
dinucleotideintheirrecognitionsite(e.g.,DraIorSspI)orthatcancleavemethylatedCpGdinucleotides(e.g.,BamHI,
KpnI,orTaqI).
Tip:Methylationpatternsdifferbetweenbacteriaandeukaryotes,sorestrictionpatternsofclonedandunclonedDNAmay
differ.
Tip:Methylationpatternsalsodifferbetweendifferenteukaryotes(seebulletsabove),affectingthechoiceofrestriction
enzymeforconstructiongenomicDNAlibraries.
Bluntended/stickyendedfragments
Somerestrictionenzymescutinthemiddleoftheirrecognitionsite,creatingbluntendedDNAfragments.However,the
majorityofenzymesmakecutsstaggeredoneachstrand,resultinginafewbasepairsofsinglestrandedDNAateachend
ofthefragment,knownasstickyends.Someenzymescreate5'overhangsandotherscreate3'overhangs.Thetypeof
digestionaffectstheeaseofdownstreamcloning:
Stickyendedfragmentscanbeeasilyligatedtootherstickyendedfragmentswithcompatiblesinglestranded
overhangs,resultinginefficientcloning.
Bluntendedfragmentsusuallyligatemuchlessefficiently,makingcloningmoredifficult.However,anybluntended
fragmentcanbeligatedtoanyother,sobluntcuttingenzymesareusedwhencompatiblestickyendedfragments
cannotbegeneratedforexample,ifthepolylinkersiteofavectordoesnotcontainanenzymesitecompatiblewith
thefragmentbeingcloned.
Compatibilityofreactionconditions
IfaDNAfragmentistobecutwithmorethanoneenzyme,bothenzymescanbeaddedtothereactionsimultaneously
providedthattheyarebothactiveinthesamebufferandatthesametemperature.Iftheenzymesdonothavecompatible
reactionconditions,itisnecessarytocarryoutonedigestion,purifythereactionproducts,andthenperformthesecond
digestion.
Restrictiondigestcomponents
Water
DNA
Buffer
Enzyme
TheamountofDNAdigesteddependsonthedownstreamapplicationandthegenomesizeoftheorganismbeing
analyzed.Werecommendusingaminimumof10gDNAperreactionforSouthernblottingofmammalianandplant
genomicDNA.FormappingofclonedDNA,0.21gDNAperreactionisadequate.
Tip:DNAshouldbefreefromcontaminantssuchasphenol,chloroform,ethanol,detergents,orsalt,asthesemayinterfere
withrestrictionendonucleaseactivity.
Oneunitofrestrictionendonucleasecompletelydigests1gofsubstrateDNAin1hour.However,supercoiledplasmid
DNAgenerallyrequiresmorethan1unit/gtobedigestedcompletely.Mostresearchersadda10foldexcessofenzymeto
theirreactionsinordertoensurecompletecleavage.
Tip:Ensurethattherestrictionenzymedoesnotexceedmorethan10%ofthetotalreactionvolumeotherwisetheglycerol
inwhichtheenzymeissuppliedmayinhibitdigestion.
Reactionvolume
Mostdigestsarecarriedoutinavolumebetween10and50l.(Reactionvolumessmallerthan10laresusceptibleto
pipettingerrors,andarenotrecommended.)
Settinguparestrictiondigestion
1.Pipetreactioncomponentsintoatubeandmixwellbypipetting.
Tip:Thoroughmixingisextremelyimportant.
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Tip:Theenzymeshouldbekeptoniceandaddedlast.
Tip:Whensettinguplargenumbersofdigests,makeareactionmastermixconsistingofwater,buffer,andenzyme,
andaliquotthisintotubescontainingtheDNAtobedigested.
2.Centrifugethetubebrieflytocollecttheliquidatthebottom.
3.Incubatethedigestinawaterbathorheatingblock,usuallyfor14hat37C.However,somerestrictionenzymes
requirehigher(e.g.,5065C)whileothersrequirelower(e.g.,25C)incubationtemperatures.
4.Forsomedownstreamapplicationsitisnecessarytoheatinactivatetheenzymeafterdigestion.Heatingthereactionto
65Cfor20minafterdigestioninactivatesthemajorityofenzymesthathaveoptimalincubationtemperatureof37C.
Notethatsomerestrictionenzymesarenotfullyinactivatedbyheattreatment.
LigationofDNA
Backtotop
InordertoconstructnewDNAmolecules,DNAmustfirstbedigestedusingrestrictionendonucleases(see Restriction
endonucleasedigestionofDNA).TheindividualcomponentsofthedesiredDNAmoleculearepurifiedandthencombined
andtreatedwithDNAligase.TheproductsoftheligationmixtureareintroducedintocompetentE.colicellsand
transformantsareidentifiedbyappropriategeneticselection.Appropriatecontrolligationsshouldalsobeperformed(see
PreparationofcompetentE.coliand TransformationofcompetentE.coli).
Removalof5'phosphatesfromlinearizedvectorDNAcanhelppreventvectorselfligationandimproveligationefficiency.
Toremove5'phosphatesfromDNA,addcalfintestinalphosphate(CIP)bufferand1UCIPandincubatefor3060minutes
at37C.Oncethereactioniscomplete,inactivateCIPbyheatingto75Cfor15minutes.
1.Atypicalligationreactionissetupasperthetable Typicalligationreaction.
2.Incubatefor124hat15C.
Tip:Simpleligationswithtwofragmentshaving4bp3'or5'overhangingendsrequiremuchlessligasethanmore
complexligationsorbluntendligations.ThequalityoftheDNAwillalsoaffecttheamountofligaseneeded.
Tip:Ligationofstickyendsisusuallycarriedoutat1215Ctomaintainabalancebetweenannealingoftheendsand
theactivityoftheenzyme.Highertemperaturesmakeannealingoftheendsdifficult,whilelowertemperaturesdiminish
ligaseactivity.
Tip:Bluntendligationsareusuallyperformedatroomtemperaturesinceannealingisnotafactor,thoughtheenzyme
isunstableabove30C.Bluntendligationsrequireabout10100timesmoreenzymethanstickyendligationsin
ordertoachieveanequalefficiency.
3.Introduce110loftheligatedproductsintocompetentE.colicellsandselectfortransformantsusingthegenetic
markerpresentonthevector(forfurtherinformation,see PreparationofcompetentE.coliand
Transformationof
competentE.coli).
4.FromindividualE.colitransformants,purifyplasmidorphageDNAsbyminiprepprocedureanddeterminetheir
structuresbyrestrictionmapping.
Tip:Itisrecommendedtoincludetwocontrolsineverytransformationexperiment:Amocktransformationwithout
DNAandatransformationwithaknownamountofclosedcircularplasmidDNA.
Typicalligationreaction
Component
Amount
ComponentDNAs
0.15g
Ligasebuffer
10mMATP
Variable
1l
T4DNAligase
20500U
Controlsareessentialifthingsgowrong.Forexample,coloniesonplatesthatreceivemocktransformedbacteriamay
indicatethatthemediumlacksthecorrectantibiotic.Anabsenceofcoloniesonplatesreceivingbacteriatransformedwith
plasmidsunderconstructioncanonlybeinterpretedifapositivecontrolusingastandardDNAhasbeenincluded.See
Bacterialcultivationmediaandantibioticsforfurtherinformationontransformationcontrols.
DNAanalysisusinganalyticalgels
Backtotop
Gelsallowseparationandidentificationofnucleicacidsbasedonchargemigration.Migrationofnucleicacidmoleculesin
anelectricfieldisdeterminedbysizeandconformation,allowingnucleicfragmentsofdifferentsizestobeseparated.
However,therelationshipbetweenthefragmentsizeandrateofmigrationisnonlinear,sincelargerfragmentshave
greaterfrictionaldragandarelessefficientatmigratingthroughthepolymer.
AgarosegelanalysisisthemostcommonlyusedmethodforanalyzingDNAfragmentsbetween0.1and25kb,whilepulse
fieldgelelectrophoresisenablesanalysisofDNAfragmentsupto10,000kb.Thissectionprovidesusefulhintsforeffective
gelanalysisofDNA.
Pouringanagarosegel
Agaroseconcentration
TheconcentrationofagaroseusedforthegeldependsprimarilyonthesizeoftheDNAfragmentstobeanalyzed.Low
agaroseconcentrationsareusedtoseparatelargeDNAfragments,whilehighagaroseconcentrationsallowresolutionof
smallDNAfragments(seetable Concentrationofagaroseusedforseparatingfragmentsofdifferentsizes).Mostgelsare
runusingstandardagarose,althoughsomespecialtypesofagaroseareavailableforparticularapplications.Forexample,
lowmeltagaroseallowsinsituenzymaticreactionsandcanthereforebeusedforpreparativegels.GenomicDNAcanbe
isolateddirectlyfromcellsimmobilizedinlowmeltagarosegels(seereference6formoreinformation).
Tip:Useultrapurequalityagarosesinceimpuritiessuchaspolysaccharides,salts,andproteinscanaffectthemigrationof
DNA.Agarosequalityisparticularlyimportantwhenrunninghighpercentageagarosegels.
Concentrationofagaroseusedforseparatingfragmentsofdifferentsizes
Agaroseconcentration(%w/v)
DNAfragmentrange(kb)
0.3*
560
0.5
130
0.7
0.812
1.0
1.2
0.510
0.47
1.5
0.23
2.0*
0.12
Adaptedfromreferences1and6.
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*Mostgelsarerunusingstandardagarose,althoughsomespecialtypesofagaroseareavailableforparticularapplications,andforvery
highorlowagaroseconcentrations.Forexample,lowmeltagaroseallowsinsituenzymaticreactionsandcanbeusedforpreparative
gels.
Electrophoresisbuffers
ThemostcommonlyusedbuffersforagarosegelelectrophoresisareTBE(TrisborateEDTA)andTAE(Trisacetate
EDTA)(seetables TAE, TBE,and Gelloadingbuffer).Althoughmorefrequentlyused,TAEhasalowerbuffering
capacitythanTBEandismoreeasilyexhaustedduringextendedelectrophoresis.TBEgivesbetterresolutionandsharper
bands,andisparticularlyrecommendedforanalyzingfragments<1kb.
ThedrawbackofTBEisthattheborateionsinthebufferformcomplexeswiththecisdiolgroupsofsugarmonomersand
polymers,makingitdifficulttoextractDNAfragmentsfromTBEgelsusingtraditionalmethods.
TAE
1xworkingsolutioncomposition
50xstocksolutioncomponentsperliter
Amountperliter
40mMTrisacetate
Trisbase
242g
1mMEDTA
Glacialaceticacid
57.1ml
0.5MEDTA,pH8.0
100ml
TBE
0.5xworkingsolutioncomposition
5xstocksolutioncomponentsperliter
Amountperliter
40mMTrisborate
1mMEDTA
Trisbase
Boricacid
54g
27.5ml
0.5MEDTA,pH8.0
20ml
Gelloadingbuffer
6xworkingsolutioncomposition
5xstocksolutioncomponentsper10ml
Amountper10ml
0.25%bromophenolblue
Bromophenolblue
25mg
0.25%xylenecyanolFF
XylenecyanolFF
25mg
40%(w/v)sucrose*
Sucrose
5ml
*15%Ficoll(Type400)or30%glycerolcanbeusedinsteadofsucrose.
Pouringthegel
1.Prepareenough1xelectrophoresisbufferbothtopourthegelandfilltheelectrophoresistank.
2.Addanappropriateamountofagarose(dependingontheconcentrationrequired)toanappropriatevolumeof
electrophoresisbuffer(dependingonthetypeofelectrophoresisapparatusbeingused)inaflaskorbottle.
Tip:Thevesselshouldnotbemorethanhalffull.Coverthevesseltominimizeevaporation.
Tip:Alwaysusethesamebatchofbuffertopreparetheagaroseastorunthegelsincesmalldifferencesinionic
strengthcanaffectmigrationofDNA.
3.Heattheslurryinamicrowaveorboilingwaterbath,swirlingthevesseloccasionally,untiltheagaroseisdissolved.
Tip:Ensurethatthelidoftheflaskisloosetoavoidbuildupofpressure.Becarefulnottolettheagarosesolutionboil
overasitbecomessuperheated.
Tip:Ifthevolumeofliquidreducesconsiderablyduringheatingduetoevaporation,makeuptotheoriginalvolume
withdistilledwater.Thiswillensurethattheagaroseconcentrationiscorrectandthatthegelandtheelectrophoresis
bufferhavethesamebuffercomposition.
4.Cooltheagaroseto5560C.
5.Pourtheagarosesolutionontothegeltraytoathicknessof35mm.Insertthecombeitherbeforeorimmediatelyafter
pouringthegel.Leavethegeltoset(3040min).
Tip:Ensurethatthereisenoughspacebetweenthebottomofthecombandtheglassplate(0.51.0mm)toallow
properformationofthewellsandavoidsampleleakage.
Tip:Makesurethattherearenoairbubblesinthegelortrappedbetweenthewells.
6.Carefullyremovethecombandadhesivetape,ifused,fromthegel.Fillthetankcontainingthegelwith
electrophoresisbuffer.
Tip:Addenoughbuffertocoverthegelwithadepthofapproximately1mmliquidabovethesurfaceofthegel.Iftoo
muchbufferisusedtheelectriccurrentwillflowthroughthebufferinsteadofthegel.
Runninganagarosegel
Backtotop
AgarosegelelectrophoresisallowsanalysisofDNAfragmentsbetween0.1and25kb(e.g.,genomicDNAdigestedwitha
frequentlycuttingrestrictionendonuclease),whilepulsefieldgelelectrophoresisenablesanalysisofDNAfragmentsupto
10,000kb(e.g.,undigestedgenomicDNAorgenomicDNAdigestedwithrarecuttingrestrictionendonucleases).The
amountofgenomicDNAloadedontoageldependsontheapplication,butingeneral,loadingoftoomuchDNAshouldbe
avoidedasthiswillresultinsmearingoftheDNAbandsonthegel.
Gelloadingbuffer(seetable Gelloadingbuffer)mustbeaddedtothesamplesbeforeloadingandservesthreemain
purposes:
Toincreasethedensityofthesamplestoensurethattheysinkintothewellsonloading.
Toaddcolortothesamplesthroughuseofdyessuchasbromophenolblueorxylenecyanol,facilitatingloading.
ToallowtrackingoftheelectrophoresisduetocomigrationofthedyeswithDNAfragmentsofaspecificsize.
MolecularweightmarkersshouldalwaysbeincludedonageltoenableanalysisofDNAfragmentsizesinthesamples.
Seetable CommonlyusedDNAmarkersinagarosegelelectrophoresisforcommonlyusedmarkers.
CommonlyusedDNAmarkersinagarosegelelectrophoresis
lHindIII
lHindIIIEcoR1
EcoR1
fX174HaeIII
21,130
21,226
21,226
1353
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9416
5184
7421
1078
6557
4973
5804
872
4361
4268
5643
603
2322
2027
3530
2027
4878
3530
310
281
564
1904
271
125
1584
234
1375
194
947
831
118
72
564
125
Preparationofsamples
1.Add1volumeofgelloadingbufferto6volumesDNAsampleandmix.
Samplesshouldalwaysbemixedwithgelloadingbufferpriortoloadingonagel.
Tip:Donotusesamplevolumesclosetothecapacityofthewells,assamplesmayspilloverintoadjacentwellsduring
loading.
Tip:Besurethatallsampleshavethesamebuffercomposition.Highsaltconcentrations,forexampleinsome
restrictionbuffers,willretardthemigrationoftheDNAfragments.
Ensurethatnoethanolispresentinthesamples,asthiswillcausesamplestofloatoutofthewellsonloading.
Agarosegelelectrophoresis
1.Applysamplesingelloadingbuffertothewellsofthegel.
Priortosampleloading,removeairbubblesfromthewellsbyrinsingthemwithelectrophoresisbuffer.
Tip:Makesurethattheentiregelissubmergedintheelectrophoresisbuffer.
Tip:Toloadsamples,insertthepipettipdeepintothewellandexpeltheliquidslowly.Takecarenottobreakthe
agarosewiththepipettip.
Tip:Oncesamplesareloaded,donotmovethegeltray/tankasthismaycausesamplestofloatoutofthewells.
Tip:Besuretoalwaysincludeatleastonelaneofappropriatemolecularweightmarkers.
2.ConnecttheelectrodessothattheDNAwillmigratetowardstheanode(positiveelectrode).
Tip:Electrophoresisapparatusshouldalwaysbecoveredtoprotectagainstelectricshocks.
3.Turnonthepowersupplyandrunthegelat110V/cmuntilthedyeshavemigratedanappropriatedistance.Thiswill
dependonthesizeofDNAbeinganalyzed,theconcentrationofagaroseinthegel,andtheseparationrequired.
Tip:AvoiduseofveryhighvoltageswhichcancausetrailingandsmearingofDNAbandsinthegel,particularlywith
highmolecularweightDNA.
Tip:Monitorthetemperatureofthebufferperiodicallyduringtherun.Ifthebufferbecomesoverheated,reducethe
voltage.
Tip:Meltingofanagarosegelduringtheelectrophoresisisasignthatthebuffermayhavebeenincorrectlyprepared
orhasbecomeexhaustedduringtherun.
Tip:Forverylongruns,e.g.,overnightruns,useapumptorecyclethebuffer.
Pulsefieldgelelectrophoresis
1.Applysamplesingelloadingbuffertothewellsofthegel.
Tip:Pulsefieldgelelectrophoresisuseshighvoltages,soTBEbuffer,whichhasgreaterbufferingcapacitythanTAE
buffer,shouldbeused.
Tip:Priortosampleloading,removeairbubblesfromthewellsbyrinsingthemwithelectrophoresisbuffer.
Tip:Makesurethattheentiregelissubmergedintherunningbuffer.
Tip:Toloadsamples,insertthepipettipdeepintothewellandexpeltheliquidslowly.Takecarenottobreakthe
agarosewiththepipettip.
Tip:Oncesamplesareloaded,donotmovethegeltray/tankasthismaycausesamplestofloatoutofthewells.
Tip:Besuretoalwaysincludeatleastonelaneofappropriatemolecularweightmarkers.
2.ConnecttheelectrodessothattheDNAwillmigratetowardstheanode(positiveelectrode).
Tip:Electrophoresisapparatusshouldalwaysbecoveredtoprotectagainstelectricshocks.
3.Turnonthepowersupplyandrunthegelat170Vwithaswitchintervalof540suntilthedyeshavemigratedan
appropriatedistance.ThiswilldependonthesizeofDNAbeinganalyzed,theconcentrationofagaroseinthegel,and
theseparationrequired.
Tip:Monitorthetemperatureofthebufferperiodicallyduringtherun.Ifthebufferbecomesheated,reducethevoltage.
Tip:Meltingofanagarosegelduringtheelectrophoresisisasignthatthebuffermayhavebeenincorrectlyprepared
orhasbecomeexhaustedduringtherun.
Tip:Forverylongruns,e.g.,overnightruns,useapumptorecyclethebuffer.
Visualanalysisofthegel
Backtotop
Staining
ToallowvisualizationoftheDNAsamples,agarosegelsarestainedwithanappropriatedye.Themostcommonlyused
dyeistheintercalatingfluorescentdyeethidiumbromide,whichcanbeaddedeitherbeforeoraftertheelectrophoresis
(seetable Comparisonofethidiumbromidestainingmethods).Alternativesincluderecentlyintroducedcommercialdyes
suchasSYBRGreen.
Tip:Stocksolutionsofethidiumbromide(generally10mg/ml)shouldbestoredat4Cinadarkbottleorbottlewrappedin
aluminumfoil.
Additionofethidiumbromidepriortoelectrophoresisaddethidiumbromideataconcentrationof0.5g/mltothe
meltedandsubsequentlycooledagarose,thatis,justbeforepouringthegel.
Mixtheagaroseethidiumbromidesolutionwelltoavoidlocalizedstaining.
Additionofethidiumbromideafterelectrophoresissoakthegelina0.5g/mlsolutionofethidiumbromide(inwater
orelectrophoresisbuffer)for3040minutes.
Tip:Rinsethegelwithbufferorwaterbeforeexaminingittoremoveexcessethidiumbromide.
Tip:Stainingbuffercanbesavedandreused.
Note:Ethidiumbromideisapowerfulmutagenandisverytoxic.Wearglovesandtakeappropriatesafetyprecautions
whenhandling.Useofnitrileglovesisrecommendedaslatexglovesmaynotprovidefullprotection.Afteruse,ethidium
bromidesolutionsshouldbedecontaminatedasdescribedincommonlyusedmanuals(1,6).
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9/12/2016
Visualization
EthidiumbromideDNAcomplexesdisplayincreasedfluorescencecomparedtothedyeinsolution.Thismeansthat
illuminationofastainedgelunderUVlight(254366nm)allowsbandsofDNAtobevisualizedagainstabackgroundof
unbounddye.ThegelimagecanberecordedbytakingaPolaroidphotographorusingageldocumentationsystem.
Tip:UVlightcandamagetheeyesandskin.AlwayswearsuitableeyeandfaceprotectionwhenworkingwithaUVlight
source.
Tip:UVlightdamagesDNA.IfDNAfragmentsaretobeextractedfromthegel,usealowerintensityUVsourceifpossible
andminimizeexposureoftheDNAtotheUVlight.
Comparisonofethidiumbromidestainingmethods
Additionofethidiumbromidepriortoelectrophoresis
Additionofethidiumbromideafterelectrophoresis
Fasterandmoreconvenientprocedure
Slowerprocedurerequiringadditionalstep
Allowsmonitoringofmigrationduringelectrophoresis
Requiresdecontaminationofgeltanksandcomb
Doesnotallowmonitoringofmigrationduring
electrophoresis
Nodecontaminationofgeltanksandcombnecessary
Moreethidiumbromideisrequired
Usuallylessethidiumbromideisrequired
ElectrophoreticmobilityoflinearDNAfragmentsisreducedby
Nointerferencewithelectrophoreticmobility
~15%
AnalysisofDNAbySouthernblotting
Backtotop
SouthernblottingisawidelyusedtechniquethatallowsanalysisofspecificDNAsequences.DNAisusuallyfirstconverted
intoconvenientlysizedfragmentsbyrestrictiondigestion.TheDNAisnextrunthroughanagarosegel(6).Southern
blotting(namedafteritsinventor,E.M.Southern)referstothetransferoftheDNAtoanylonornitrocellulosemembraneby
capillarytransfer.TheDNAofinterestcanbeidentifiedbyhybridizationtoradioactiveorchemiluminescentprobesand
visualizedbyautoradiographyorstaining.
ManyvariationsontheSouthernblottingprocedureexist.Astandardprotocolisdescribedheretogetherwithrecipesfor
buffersandsolutions.
Equipmentrequired
Whatman3MMfilterpaper
Blottingmembrane
Papertowels,astackofapproximately1520cm
Plasticwrap
TwoglassorPlexiglasplates
Buffertray(e.g.,glasscasseroledish)capableofholding12litersofbuffer
Support(tobeplacedinthebuffertray)
Flatweight,approximately1kg
Oven,at80C,orUVtransilluminator
Orbitalshaker
PreparationofgelsforSouthernblotting
FragmentationoflargeDNAmolecules(optional)
DNAfragmentslongerthan10kbdonottransfertoblottingmembranesefficiently.Inordertofacilitatetheirtransfer,these
fragmentsarereducedinsize,eitherbyaciddepurinationorbyUVirradiation.
Aciddepurinationimmediatelyaftergelelectrophoresis,placethegelinasolutionof0.2MHCl,sothatitiscompletely
covered.Agitategentlyfor10minutes.Duringthisperiodthecolorofthebromophenolblueinthesampleswillchange
frombluetoyellow,indicatingthatthegelhasbeencompletelysaturatedwiththeacid.Rinsethegelbrieflyindistilled
water.
Tip:Thedepurinationstepshouldnotlasttoolong,sinceveryshortfragmentsattachlessfirmlytothemembrane.
Tip:Depurinatedgelsmayyieldfuzzybandsonthefinalautoradiograph,presumablybecauseofincreaseddiffusionof
theDNAduringtransfer.Depurinationisthereforerecommendedonlywhenfragmentslargerthan10kbaretobe
transferred.
UVirradiationexposethegeltoUVlightatawavelengthof254nmfromasourceoperatingat30W,for3060
seconds.
Denaturation
DoublestrandedDNAmustbedenaturedinordertocreatesuitablehybridizationtargets.Completelycoverthegelwith
denaturationbuffer(seetable Denaturationbuffer)andincubatefor30minuteswithgentleshaking.Ifaciddepurination
wasusedtodenaturetheDNA,thebromophenolbluewillreturntoitsoriginalcolorduringthisincubation.
Neutralization
Removethedenaturationbufferandcompletelycoverthegelinneutralizationbuffer(seetable Neutralizationbuffer).
Incubatefor30minuteswithgentleshaking.
Denaturationbuffer
Compositionofworkingsolution
Component
Amountperliter
1.5MNaCl
NaCl
87.7g
0.5MNaOH
NaOH
20g
Component
Amountperliter
1MTrisCl
Trisbase
121.1g
1.5MNaCl
NaCl
87.7g
Neutralizationbuffer
17/19
9/12/2016
BuffershouldbepH7.4.AdjusttopH7.4withHCl.
20xSSC
Component
Amountperliter
3MNaCl
NaCl
175.3g
0.3MSodiumcitrate
SodiumcitrateH2 O
88.2g
AdjusttopH7.4withNaOH.
Assemblingtheblottingapparatus
1.Placeasupportlargerthanthegelinatraycontaining10xSSC(seetable 20xSSC),andcoverthesupportwitha
glassorPlexiglasplate(seefigure Southernblotsetup).
2.CuttwolengthsofWhatman3MMpaperwiderthanthegel,longenoughtofitunderthegelandreachtothebottomof
thedishoneitherside.Wetthesheetsbrieflyin10xSSC,andplacethemontheglassplate.Removeanyairbubbles
betweenthepaperandthesupportbyrollingapipetseveraltimesbackandforthoverthesurface.
3.CutonesheetofblottingmembraneandtwosheetsofWhatman3MMpaperabout1mmlargerthanthegeloneach
side.
Tip:Alwaysweargloveswhenworkingwithblottingmembranes.Handlemembranescarefullybytheedgesorusing
cleanbluntendedforceps.
4.Placethepreparedgelupsidedownontheplatform.Removeanyairbubblestrappedbetweenthegelandthe
platformbyrollingapipetseveraltimesbackandforthoverthegel.
5.Surroundthegelwithplasticwrap.Thisensuresthatthe10xSSCmovesonlythroughthegelandnotaroundit.
6.Placetheprecutblottingmembraneontopofthegelsothatitcoverstheentiresurface.Donotmovetheblotting
membraneonceithasbeenplacedonthegel.Removeanyairbubblesbetweenthepaperandthesupportas
describedinstep4.
7.BrieflywetthetwoprecutsheetsofWhatman3MMpaperin10xSSC,andplacethemontopofthenylonmembrane.
Again,removeanytrappedairbubblesasdescribedinstep4.
8.Placea1520cmstackofdrypapertowelsontopofthefilterpaper.
Tip:Makesurethattheplasticwrapsurroundingthegelpreventscontactofthepapertowelswiththe10xSSCandthe
wetfilterpaperunderthegel.Ensurethatthetowelsdonotdroopoversincetheycancauseliquidtoflowaroundthe
gelinsteadofthroughit.
9.PlaceasecondglassorPlexiglasplateontopofthepapertowels.Placetheweightontopoftheplate.Letthetransfer
proceedfor1218h.
Tip:Transferefficiencyisimprovedbyremovingthewetpapertowelsandreplacingthemwithdryonesatleastonce
duringthetransfer.
FixingtheDNAtotheblot
1.Afterthetransferiscomplete,removetheweight,papertowels,andthetwosheetsoffilterpaper.Turnoverthegeland
theblottingmembranetogether,andlaythem,gelsideup,onasheetofdryfilterpaper.Markthepositionsofthegel
lanesonthemembraneusingaballpointpenorasoftleadpencil.Peelthegelfromthemembrane.Ifdesired,keep
thegeltoassesstheefficiencyofDNAtransfer,otherwisediscard.
Beforeremovingthegelfromtheblottingmembrane,ensurethatthegellanesaremarkedsothattheycanbelater
identified.
InordertoassesstheefficiencyofDNAtransfer,stainthegelwithethidiumbromideafterblottingtoseehowmuch
DNAremains.
2.FixtheDNAtotheblot,eitherbybaking(seestep3)orbyUVcrosslinking(seestep4).
Tip:UVcrosslinkinggenerallygivesbetterresultsandenhancedsensitivitycomparedwithbaking.However,effective
crosslinkingrequiresoptimizationofthesystem.
3.Useeitherthissteporstep4:TofixtheDNAtothemembranebybaking,firstlettheblotairdryonasheetoffilter
paper,thenplacebetweentwosheetsoffilterpaper,andbakeat80Cfor2h.Proceedtostep5.
4.Useeitherthissteporstep3:TofixtheDNAbyUVcrosslinking,firstprotectthesurfaceofthemembranebycovering
theUVsource(e.g.,atransilluminator)withplasticwrap.ThentakethedampblotandexposethesidewithDNAtothe
UVsourceforapredeterminedlengthoftime.Proceedtostep5.
Tip:ItisimportanttooptimizethesystemforUVcrosslinking.Todothis,prepareablotwithseveralcontrolDNA
samples.Cuttheblotintoseparatestripsforeachlane,andirradiateeachblotfordifferenttimes,varyingfrom0.5to5
min.Hybridizealltheblotstogetheranddeterminewhichtimegivestheoptimalsignalintensity.Itisimportanttouse
thesameconditions(UVwavelength,distancefromUVsource)foreachexperiment.Itisalsoimportanttocalibratethe
systemroutinely,astheenergyemittedfromaUVbulbisreducedwithuse.
Tip:UVlightcandamagetheeyesandskin.Alwayswearsuitableeyeandfaceprotection.
5.Iftheblotwillnotbeusedimmediately,storeitatroomtemperaturecoveredinplasticwrap.
DNAcleanup
Backtotop
CleanupofDNAisoftenaprerequisiteforefficientdownstreamapplicationssuchascloning,sequencing,microarray
analysis,oramplification.
Theuseofadedicatedkitforthisapplicationmaybenecessary,sincekitscanvarydependingonthetypeofreactionand
DNAfragmentsize(e.g.,PCRproducts,gelextraction,enzymaticreactions,nucleotideremoval,dyeterminatorremoval)
andtherequiredelutionvolume.
StandardPCRcleanuponlyrequirestheremovalof~20boligos.Innextgenerationsequencing(NGS)librarypreparation,
often,muchlargerprimersalmostintherangeofaPCRampliconmustberemoved,andPCRcleanupmaynotbe
sufficient.Forthisspecializedsizeselectionprocedures,dedicatedkitsorPEGbasedprecipitation(8)arenecessary.
References
Backtotop
1.Ausubel,F.M.,etal.(1991)CurrentProtocolsinMolecularBiology.NewYork:JohnWileyandSons.
2.AnimalGenomeSizeDatabasewww.genomesize.com.
3.Systma,K.K.,Givnish,T.J.,Smith,J.F.,andHahn,W.J.(1993)Collectionandstorageoflandplantsamplesfor
macromolecularcomparisons.MethodsEnzymol.234,23.
4.Birnboim,H.C.,andDoly,J.(1979)ArapidalkalinelysisprocedureforscreeningrecombinantplasmidDNA.Nucl.
Acids.Res.7,1513.
5.Birnboim,H.C.(1983)ArapidalkalineextractionmethodfortheisolationofplasmidDNA.MethodsEnzymol.100,243.
6.Sambrook,J.,Fritsch,E.F.,andManiatis,T.(2012)MolecularCloning:ALaboratoryManual.4thed.ColdSpring
Harbor,NY:ColdSpringHarborLaboratory.
7.Wilfinger,W.W.,Mackey,M.,andChomczynski,P.(1997)EffectofpHandionicstrengthonthespectrophotometric
assessmentofnucleicacidpurity.BioTechniques22,474.
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8.Lis,T.,andSchleif,R.(1975)SizefractionationofdoublestrandedDNAbyprecipitationwithpolyethyleneglycol.
NucleicAcidsRes.2,383.
Backtotop
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Backtotop
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