AFLP protocol.docx

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