AFLP protocol.docx
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AFLPprotocol
AFLPprotocol
SamuelHazen
MichiganStateUniversity
WheatBreedingandGenetics
RichardW.Ward-WheatBreeder
ThisprotocolisanamalgamationofprotocolsusedandprovidedbyVosetal.,BrentBarrett(WSU),GregPenner(AgCA),A.S.Reddy(TexasA&M),GibcoBRL,andothersaswellasadditionsandchangesmadeinWheatlab.
I.RestrictionDigestionofGenomicDNA
Starting[]
Final[]
Perrxn(µl)
OnePhorAll(OPA,Pharmacia)
10X
1X
5.0
MseI(NewEnglandBiolabs)
4U/µl
5U
1.25
EcoRI(GibcoBRL)orPstI(Promega)
10U/µl
5U
0.5
BSA(comesw/MseI)
10µg/µl
5µg
0.5
ddH2O
32.75
GenomicDNA
50-250ng/µl
10µl
Total
50µl
1.Heatoneovento700andtheotherto370.
2.Distribute40µlofcocktailineachlabeledtube.
3.Add10µlofDNAtoeachtube.
4.Vortexandbrieflycentrifuge.
5.Incubate@370for3hours.Agitateeveryhourorso.Idoaquickvortexsothesamplestaysinbottomoftubeandcentrifugationisnotneeded.
6.Inactivateenzyme@700for15min.
II.Adapterpreparation
Completeduringorbeforedigestion
EcoRIAdapter(120ligationrecipe)
EcoRI.1oligo(1µg/µl)
3.4µl
EcoRI.2oligo(1µg/µl)
3.0µl
OPA
6.0µl
ddH2O
107.6µl
MseIAdapter(120ligationrecipe)*
MseI.1oligo(0.5µg/µl)
64.0µl
MseI.2oligo(0.5µg/µl)
56.0µl
OPA
7.0µl
*MseIoligosmayneedtobespeedvacuumedinordertoincreaseconcentration.
Mixinthermocyclertubesandrunfile#44.
650Cfor10min.
370Cfor10min.
250Cfor10min.
Storeat-200C.
III.LigationofAdapters.
Perrxn
EcoRIadapter
1µl
MseIadapter
1µl
T4DNAligase10Xbuffer
1µl
T4DNAligase(3U/µl,Promega)
0.33µl
ddH2O
6.7µl
1.Add10ulofligationmixto50ulofdigestedDNA.Vortexandbrieflycentrifuge.
2.Incubateatroomtemperaturefor3hrs.Agitateeveryhourorsoasabove.
IV.PreamplificationReactions
EcoRI+Aoligo(50ng/µl)
0.5µl
MseI+Coligo(50ng/µl)
0.5µl
dNTPs(5mM,Gibcoas100mM)
2µl
10XPCRbuffer(w/Taq)
2µl
Taqpolymerase(5U/µl,Promega)
0.1µl
MgCl2(w/Taq)
1.2µl
ddH2O
11.9µl
TemplateDNAfromrestriction/ligation
2µl
Thermocyclerfile#36
94
2min
94
1min
26cycles
56
1min.
72
1min.
72
5min.
4
hold
1.TransferPCRproductintonewtubeswith100µlsterileddH2O.
2.Blottestingcantestreactionsuccess.Dot2µlofEthidiumbromide(2µg/µl)and3µlofproductonplexi-glass.Use3µlofcocktailascontrol.VisualizedotsusingUVbox.
V.Selectiveamplification
24
36
48
EcoRI+ANNoligo(50ng/µl)
0.5µl
13
19.5
26
MseI+CNNoligo(50ng/µl)
0.6µl
15.6
23.4
31.2
dNTPs(5mM,Gibcoas100mM)
0.8µl
20.8
31.2
41.6
10XPCRbuffer(w/Taq)
2.0µl
52
78
1.4
Taqpolymerase(5U/µl,Promega)
0.08µl
2.1
3.12
4.16
MgCl2(w/Taq)
1.2µl
31.2
46.8
62.4
ddH2O
13.82µl
360
540
720
DilutetemplateDNAfrompre-selectivePCR
1µl
Thermocyclerfile#20
94
2min
94
30s
12cycles,decreaseannealingtempby0.7each
65
30s
72
1min.
94
30s
23cycles
56
30s
72
1min.
72
2min.
4
hold
3.Testproductusingdotblotifnecessary.
4.Combine8µlformamide-loadingbufferandPCRproduct.
VI.Gelelectrophoresis
1.Acrylamidegelsolution
42gUrea
10ml10xTBE
15ml40%acrylamide
waterupto100mls
2.Combineurea,TBE,andapprox.25mlwaterinabeaker.Stirwithheatuntilureadissolves.
3.Transfersolutionto100ml-graduatedcylinderandaddwaterupto85ml.Transfertovacuumflask.
4.Addacrylamidetoflaskanddegasforapprox.10min.
5.Transfersolutiontobeakerandadd100µlTEMEDand500µl10%freshAPS.Drawsolutionintosyringe.Keeptipsubmergedatalltimes.
6.Placetubeonsyringeandturnitupward.Pushairoutoftubeandpinchendoftube.InsertintoCasterbase.
7.Glasspreparation(allglassmustbescrupulouslyclean!
)
8.WipeIPCunitwithchem-wipeandethanol.
9.GlassshouldbetreatedwithSigmacoteaboutevery5gelsrunoruntiltopofgelstickstolongglass.Saturateachem-wipewithSigmacoteandwipeverticallyandhorizontally.Waitfiveminutesandwipeglassthreetimeswithethanol.Changegloves.
10.Wipelongglasswithethanolandchem-wipe.
11.InanEppindorftubecombine1mlof95%EtOH0.05%Aceticacidand2µlofbindsilane.
12.TreatglasssameasabovedescriptionofSigmacote.UseagreatdealofpressurewhenwipingwithEtOH.Changegloves.
13.IntheeventofacontaminationofeitherSigmacoteorBindsilaneontherespectiveglass,soakin10%NaOH.
14.Whilehorizontal,placespacersonIPCandlongglassontop.
15.Erectverticallyandclampsidebraces.
16.AttachCasterbaseinsertpegsandturn.BesuretodothiswhileverticalandthatyoucanseethespaceinbetweenglassplatesthroughCasterbasehole.
17.Checktoseeifcombwilleasilyinsertbetweenglass.Ifnot,adjust.
18.Leanclampsontopoftuberacks.
19.Injectgelsolution.
20.Insertcombsandadjustunittohorizontalposition.
21.Allowgeltopolymerizeforatleast1hour.
VII.Gelloading
1.Fillbottomtraywith1XTBEsoabout½inchofthebottomofgelunitissubmerged.FillIPCuntil½inchaboveshortglass.Useneedleandflushoutwell
2.Rungelat75Wfor1hr.
3.Flushwellagainandinsertcombwithoutpiercinggel.
4.Load4.5µlsample.
5.Rungelfor10minandthenremovecomb(goodtimetomakefix/stopanddevelopingsolution).
6.Rungelfortotalof2hrand50min.(Lightbluedyeshouldmigrate1inchbelowbottomribofIPC.
7.InserttubeinIPCanddrainbuffer.
8.PullglassapartandwashIPC.
VIIISilverStaining
FromPromegaTechnicalManualandSamandSuzanneDowneyempiricalknowledge.
1.Separateplateswhilekeepingthegelattachedtoshortglass.
2.Fixthegel:
Placegelintray,coverwithcoldfix/stopsolutionandagitatewellfor20minutes.Gelmaybestoredinfix/stopsolutionovernight.Savefix/stopsolutionandplacebackinfreezer.
3.Washthegel:
Rinsethegel3timesfor2-3min.eachinddH2Ousingagitation.Liftgelfromsolutionandallowtodrain10-20seconds.
4.Stainthegel:
Transferthegeltostainingsolutionandagitatewellfor30minutes.
5.Pour1Lofthedevelopingsolutionintoatray.Transferstainingsolutiontobeaker.RinsetrayandfillwithddH2O.
6.Rinsegelfor5-10secondsONLY.Transfertodevelopingsolution.
7.Agitateindevelopingsolutionuntilbandsbegintoappear.Transfergeltoremainingchilleddevelopingsolutionfor2-3minutes.
8.Fixthegel:
add1LofFix/stopsolutiondirectlytodevelopingsolutionandagitatefor2-3minutes
9.RinsegeltwicefortwominuteseachinddH2O.
10.Drygelonglass
Fix/stopsolutionStainingsolution
200mlofglacialacidicacid2g(1packet)ofsilvernitrate(AgNO3)
1,800mlultrapurewater3ml(1vial)of37%Formaldehyde
Freezeforapprox.3hours2Lultrapurewater
Developingsolution
60g(1packet)SodiumCarbonate(Na2CO3)
2Lultrapurewater
**chillto10oC.Iplacesol.Infreezerforapprox4hoursandstirtobreakupicepriortouse.
Immediatelybeforeuseadd
3ml(1vial)of37%Formaldehyde
400μlaliquotSodiumThiosulfate(discardremaining)
IX.Gelscoringandscanning.
1.Scangelintwosectionswithouttwooptionsselected.Saveascompressedtifandjpg.
2.Scoregelwhilestillonglassandmakenotesonprintoutofscannedimage.
3.Keeporiginalscoresheetandgelprintoutinfolder
4.Recordgelinrecordformwithallpertinentdetails.
X.Gelpreservation
1.Soakgelin3%NaOHwithgentleagitationfor30to60min,oruntiledgeofcornerofthegelstartscomingloose.Ifgeldoesnotcomeloose,teaseacornerandpullgently.Ifitpeelseasily,gelisreadyfortransfer.Loosenedgeswithrazorbladetofacilitatetransfer.
2.Carefullytransfergelto3.5%aceticacidandsoakfor3minwithoutagitation.RinseinddH2Ofor2minuteswithoutagitation.
3.Drainexcesswaterfromgelandsmoothasheetofchromatographypaperovergel.
4.Veryslowlypulledgeorcornerupwhilegeladherestopaper.Usearazorbladetopersuadeanylaggingpartsofthegel.
5.Covergelwithplasticwrapanddryongeldryerat70ofor2hrs.
OligoFragments
EcoRILinker1
CTCGTAGACTGCGTACC
EcoRILinker2
AATTGGTACGCAGTCTAC
EcoRI+A
GACTGCGTACCAATTCA
PstILinker1
CTCGTAGACTGCGTACATGCA
PstILinker2
TGTACGCAGTCTAC
PstI+A
GACTGCGTACATGCAGACA
MseILinker1
GACGATGAGTCCTGAG
MseILinker1
TACTCAGGACTCAT
MseI+C
GATGAGTCCTGAGTAAC