gel electrophoresis - mcdb凝胶电泳- mcdb

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1、HC70ALSPRING2004PROFESSORBOBGOLDBERGGELELECTROPHORESISReference:Sambrooketal.(1989).MolecularCloningManual.SolutionsNeeded:ØAgaroseØ1XTAEbufferØ10mg/mLEthidiumBromide(EtBr)Ø6XLoadingDyecontainingxylenecyanolandbromophenolbluedyesApparatusNeeded:ØGelcast

2、ØGelboxØCablesØGelcombØPlastic(Saran)wrapHC70ALSPRING2004PROFESSORBOBGOLDBERGGELELECTROPHORESIS1.MeasureoutXgramsofagarose(powder)dependingonthefinalpercentageofagaroseinthegel.Example:Ifyouwanttomakea1%agarosegel(1g/100mL,w/v),weighout1gofagarosefor100

3、mLofagarosesolution2.Carefully,puttheagaroseina250-mLErlenmeyerflask.3.Measureout100mLof1XTAEbufferusingaplasticorglassgraduatedcylinder.4.Add100mLof1XTAEbufferintotheflaskinstep2.5.Covertheflaskwithapieceofplasticwrap.Poke3-4holesontheplasticwrapusinga

4、pointedendofapencilorpen(note:theholesallowthesteamtoescapeduringmicrowavinginstep6below).Swirlthesolutiontobreakupanylumpsofagarosegranules.6.Microwavethesolutionforabout2minutesoruntiltheagarosegranuleshavecompletelymelted.ØBecarefulwiththeflask.Theso

5、lutiongetsveryhot.ØConstantlywatchoverthesolutionbecausewhenitstartsboiling,itmightoverflow.ØSwirlgentlythesolutionseveraltimeswhilemicrowavingtohelpmeltagaroseevenly.ØOncetheagarosehasmeltedcompletely,thesolutionisclear.ØOncetheagarosehasmeltedcomplete

6、ly,thesolutionisclear.7.Cooldowntheagarosesolutionforatleast30minina55ºCwaterbath.8.Whiletheagarosesolutioniscooling,preparethegelcastwiththeappropriatecomb.ØThecombdependsonthenumberofPCRsamples.Forexample,ifthereare31samples,thena40-toothcombisneeded.

7、ØRemembertoaddtwomorewellstothenumberofwellsneededforthesamples.Thesetwowellswillbeforloading1kbDNAladderinthefirstandthelastwells.HC70ALSPRING2004PROFESSORBOBGOLDBERG1.Add2mLofloadingdyeinnewlylabeledmicrocentrifugetubesforeachPCRsample.Thetubesarelabe

8、ledaccordingtothesamples.2.Aliquot10mLofPCRsampleintomicrocentrifugetubes.3.Aftertheagarosesolutionhasbeencooleddown,addtoit5mLofEthidiumBromide(EtBr)andswirltomix.4.Pourtheagarose/EtBrsolutionintothegelcast.Waitfor30minfortheaga