VectorettePCRofYeastDNA

CarlFriddle

1)Cut1-3µgofcleanDNAovernightwith8-10Uofbluntcuttingenzymein20µl

Mostproblemscomefromdirty,uncutDNA.Phenol/glassbead/RNasepreparedDNAworkswell

RsaI,AluIandDraIprovidegoodresults.

2)Heatinactivateenzyme

3)Add:

3µl10xNEBufferusedindigest
1µlannealedanchorbubble
1µl(400U)ligase
0.5µlof5mMATP(50µMATPfinal)
25.5µlWater

4)Incubateat16Cfor9-24hours.

5)Use5µlin100µlPCR.PerkinElmerAmpliwaxisrecommendedforhotstart.

5µlofligation
2.5µlof20µMspecificprimer[M13(-47)formTn3library]
2.5µlof20µM224primer
8µlof2.5mMdNTPs
10µlofTaqPCRbuffer
71µlWater
1µlTaqDNApolymerase(5U)
TransfertoPerkinElmer9600ThermalCycler
- Denature92C,2minutes
- 35Cycles[92C,20sec;67C,30sec;72C,45-180sec(>1min/1kb)]
- 72C,90sec

6)Gelpurify80µlofPCRproductin1-3%SeaKemGTG,extractwithQiaex(Qiagen),elutewith12µlofddWater

7)Sequence7µlwithSequenasekitfromAmersham.

Use1µlof200-600µMspecificprimer[M13(-47)formTn3].Undiluted10ODsynthesisfromGensetworkswell.

UsehighspecificactivityS-35(>1000Ci/mmol,AmershamAG1000)

Boil10"andfastcoolinicewater.

AnchorBubbleprimers

3"GAGAGGGAAGAGAGCAGGCAAGGAATGGAAGCTGTCTGTCGCAGGAGAGGAAG5"||||||||||||||||||||||||||||||||||||5"GACTCTCCCTTCTCGAATCGTAACCGTTCGTACGAGAATCGCTGTCCTCTCCTTC3"PRIMER2245"CGAATCGTAACCGTTCGTACGAGAATCGCT3"

Toannealbubbleprimers,heata2-4µM(inddWater)to65Cfor5minutes,thenaddMgCl₂to1-2mMandallowtocooltoroomtemperature.

Wouldyouliketoseeadiagram?

References:

Rileyetal,NucleicAcidsResearch18:2887-2890,1990
Footeetal,Science258:60-66,1992
Burnsetal,GenesDev.8(9):1087-1105,1994
Vollrath,LargeDNACourse,ColdSpringHarborLaboratory,1995

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