Thefollowingprotocolisdesignedforsubcloninginserts(I)fromonevectorintoanothervector(V).Theinsertscanbeanywherefrom30bpto8kb(possIBLyhigher).

Performrestrictiondigestsof(I)and(V)toobtaincompatibleoverhangs(evenbluntligationsworkwellwiththisprotocol).Generally,performrestrictiondigestsasfollows:

DNA1-10ul(2ugTOTAL)

10XBuffer5ul(determineappropriatebufferfromNEBcatalog)

Enzyme11-2ul(20-40Uissufficient)

Enzyme21-2ul(sameasabove)

100XBSA1ul

Waterto50ultotalvolume

Note:AddenzymesLAST.Digestat37Cfor2-3h(nolonger!).

1.OPTIONAL:tothe(V)digest,add1ulofCalfIntestinalAlkalinePhosphatase(CIP)—BoehringerMannheim,1U/ul.IncubateatROOMTEMPforanadditional30min.

2.Duringtheincubation,preparea1-2%agarosegelin1XTAE(NOTTBE!)withwidelanes(8-10mmwide).DONOTaddEtBrtotheagarosegel(otherwisethiswillmutateyourDNAsamples).

3.Add10ulof6XagarosegelloADIngbuffertothedigests,andload30ulofthedigestsintoalaneintheagarosegel.Load7ulof1kbDNAladderinaseparatelane.

4.Runthegelat70Vfor30minto1h.

5.Stainthegelina2.5ug/mlsolutionofEtBrfor5-10min(higherthepercentagegel,thelongerthestaintime).

6.Runthegelagainat70Vfor10min(thiswillrapidlydestainthegel).

7.ViewthegelatLOWINTENSITYontheUVgelbox,andtakea2.5sec.Exposurepictureforyourrecords.MinimizeexposureofthegeltoUVlight.

8.UsingaCLEANbladeforeachfragment,excisethedesiredfragmentswithaslittleexcessagaroseaspossible,andplaceinapreweighedandlabeledEppendorftube.

9.Weightheagarosegelslice,andadd3volumesofbufferQG(fromtheQiagenGelExtractionKit).Incubateat42Cfor10-15minuntilthegelsliceiscompletelydissolved.

10.Add1volumeofisopropanol,mix,andloadonaPURPLEspincolumn.Spinattopspeedfor30sec.Anddiscardtheeluate.

11.Washthecolumnwith500ulofbufferQG,spin,thenwashwith750ulofPE.

12.SpinafinaltimetoremovealltracesofPE.

13.ElutetheDNAwith50ulofWATER.

ProceedtoLIGATION:

LIGATION:

14.BoehringerMannheimRapidLigationKit.ThawDNAdilutionbufferandT4ligasebufferquicklyandplaceonice.

15.Add2ulof(V)and2ulof(I)to1ulof5XDNAdilutionbufferinaneppendorftube.

16.Add5ulof2XLigaseBuffer,flicktomix,andadd0.5ulofLigase.Mixandincubateatroomtempfor5min.

17.Placeonice,andproceedtoTRANSFORMATION.

TRANSFORMATION:

18.QuicklythawatubeofDH5aandplaceonice.Prechillanemptyeppendorfonice,andadd90uloftheDH5aintothistube.

19.Add8uloftheligationreactiontotheDH5a,andincubateonicefor30min.

20.Heatshockat42Cfor45sec.

21.Coldshockonicefor2min.

22.Add900ulofLBandincubateat37Cwithshakingfor30min.

23.Spintubeat5Krpmfor2min.Removeallbut100uloftheLBandresUSPendpellet.

24.PlatepelletonLBplatewithappropriateantibiotic.

25.Incubateinvertedat37Covernight