AFLP protocolWord文件下载.docx

AFLP protocolWord文件下载.docx

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10U/µ

0.5

BSA（comesw/MseI）

10µ

g/µ

5µ

g

ddH2O

32.75

GenomicDNA

50-250ng/µ

Total

50µ

1.Heatoneovento700andtheotherto370.

2.Distribute40µ

lofcocktailineachlabeledtube.

3.Add10µ

lofDNAtoeachtube.

4.Vortexandbrieflycentrifuge.

5.Incubate@370for3hours.Agitateeveryhourorso.Idoaquickvortexsothesamplestaysinbottomoftubeandcentrifugationisnotneeded.

6.Inactivateenzyme@700for15min.

II.Adapterpreparation

Completeduringorbeforedigestion

EcoRIAdapter（120ligationrecipe）

EcoRI.1oligo（1µ

3.4µ

EcoRI.2oligo（1µ

3.0µ

OPA 

6.0µ

107.6µ

MseIAdapter（120ligationrecipe）*

MseI.1oligo（0.5µ

64.0µ

MseI.2oligo（0.5µ

56.0µ

7.0µ

*MseIoligosmayneedtobespeedvacuumedinordertoincreaseconcentration.

Mixinthermocyclertubesandrunfile#44.

650Cfor10min.

370Cfor10min.

250Cfor10min.

Storeat-200C.

III.LigationofAdapters.

Perrxn

EcoRIadapter

1µ

MseIadapter

T4DNAligase10Xbuffer

T4DNAligase（3U/µ

l,Promega）

0.33µ

6.7µ

1.Add10ulofligationmixto50ulofdigestedDNA.Vortexandbrieflycentrifuge.

2.Incubateatroomtemperaturefor3hrs.Agitateeveryhourorsoasabove.

IV.PreamplificationReactions

EcoRI+Aoligo（50ng/µ

0.5µ

MseI+Coligo（50ng/µ

dNTPs（5mM,Gibcoas100mM） 

2µ

10XPCRbuffer（w/Taq）

Taqpolymerase（5U/µ

l,Promega）

0.1µ

MgCl2（w/Taq）

1.2µ

11.9µ

TemplateDNAfromrestriction/ligation

Thermocyclerfile#36

94

2min

1min

26cycles

56

1min.

72

5min.

4

hold

1.TransferPCRproductintonewtubeswith100µ

lsterileddH2O.

2.Blottestingcantestreactionsuccess.Dot2µ

lofEthidiumbromide（2µ

l）and3µ

lofproductonplexi-glass.Use3µ

lofcocktailascontrol.VisualizedotsusingUVbox.

V.Selectiveamplification

24

36

48

EcoRI+ANNoligo（50ng/µ

13

19.5

26

MseI+CNNoligo（50ng/µ

0.6µ

15.6

23.4

31.2

0.8µ

20.8

41.6

2.0µ

52

78

1.4

0.08µ

2.1

3.12

4.16

46.8

62.4

13.82µ

360

540

720

DilutetemplateDNAfrompre-selectivePCR

Thermocyclerfile#20

30s

12cycles,decreaseannealingtempby0.7each

65

23cycles

2min.

3.Testproductusingdotblotifnecessary.

4.Combine8µ

lformamide-loadingbufferandPCRproduct.

VI.Gelelectrophoresis

1.Acrylamidegelsolution

42gUrea

10ml10xTBE

15ml40%acrylamide

waterupto100mls

2.Combineurea,TBE,andapprox.25mlwaterinabeaker.Stirwithheatuntilureadissolves.

3.Transfersolutionto100ml-graduatedcylinderandaddwaterupto85ml.Transfertovacuumflask.

4.Addacrylamidetoflaskanddegasforapprox.10min.

5.Transfersolutiontobeakerandadd100µ

lTEMEDand500µ

l10%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%Aceticacidand2µ

lofbindsilane.

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.Fillbottomtraywith1XTBEsoabout½

inchofthebottomofgelunitissubmerged.FillIPCuntil½

inchaboveshortglass.Useneedleandflushoutwell

2.Rungelat75Wfor1hr.

3.Flushwellagainandinsertcombwithoutpiercinggel.

4.Load4.5µ

lsample.

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

TACTCAGGACTCAT

MseI+C

GATGAGTCCTGAGTAAC