Plasmid preparation without overnight culutre
来自 : 蚂蚁淘
PlasmidpreparationwithoutovernightculutreContributedbyDr.A.Gratchev- Prepare13mltubewith5mlLB,containingappropriateantibiotic(ampicillinconcentrationcanbeupto400µg/ml).
- Pickupthecolonywithsterileyellowpipetttip,dropthetipinthetube
- ShakeyourtubersattherotaryshakerasfastaspossIBLe(usually300-400rpm)at37°C.Highertemperatures(upto42°C)canbeusedforsomeplasmidsandE.colistrains.
- Mostofthecultureswillshowsufficientdensityafter8-10hincubation.
- Transfer1.5mlofculturein1.5mltubeandcentrifugeintabletopcentrifugeatmaxspeedfor1-2min.
- Discardthesupernatant.
- Ifthepelletissmallrepeat2previoussteps.
- Add100µlofP1buffer(Qiagen)andvortexuntilthepelletiscompletelyresUSPended.
- Add100µlofP2buffer(Qiagen)andmixbyshakinginupside-downposition.
- Add100µlofP3Buffer(Qiagen)andmixverycarefully.
- Centrifugeat13000rpmfor15min.Whilecentrifugingprepareasetoffresh1.5mltubes.
- Transferthesupernatantintoafreshtube,trynottotakedebris.
- Add900µl100%EtOH,vortexbriefly.
- Centrifuge15minat13000rpm.
- DiscardthesupernatantandwashDNAPelletwith500µl70%Ethanol.
- Centrifuge2minat13000rpm.
- DissolvetheDNApelletin50µlofpureH2O.
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