E.coli Competent Cells DH5α
来自 : 蚂蚁淘
| FROZENCOMPETENT E.COLICELLS |
(Inoueetal.,1990Gene96:23)- Inoculatea5mlovernightofE.coliinLB+20mMMgSO4.
- Nextmorning,inoculate250mlLB+20mMMg++ina2Lflaskwithabout2mlovernightculture.Growatroomtemp(23°C)withgoodaeration(250rpm)toanA600of0.4-0.6.Tempisimportant--seeoriginalref.
- Placecells10minonice.Transfertoasterilebottleandspin3K,10",4°C.
- ResUSPendpelletin80mlcoldTB(swirlcellsinbottle).Leave10’/ice.
- Spincells3K,10",4°C.
- Resuspendcellsin20mlcoldTBthenadd1.5mlDMSO.Leave10"/ice.
- Dispenseinto220uland525ulaliquots(incoldsteriletubes)andfreezeindryice/EtOHbath.Store-70°C.Typically,competencyabout5X106cfu/ugDNA.Note,improvesafterfreezing.Cellsgoodforayearandcounting.
Touse: - To50or100ulcells,add5-50ngDNA.Leave30’onice.
- Heatshock,45secat42C,thenchillcellsoniceabout2’.
- Spindown(15sec,Eppendorffuge)andremoveSN.(RemovingtheManganeseseemstoboostefficiencyabout10X)andresuspendcellsin200ulLB.
- Forasupercoiledplasmid,plate1ulofcells.Foraligation,plate20ulandtherest.
TB(transformationbuffer:filtersterilizeandstore4°C) | Product | [stock] | []final | volumestomake100ml | | Pipes-NaOHpH6.7 | 0.5M | 10mM | 2ml | | CaCl2 | 0.5M | 15mM | 3ml | | KCl | 2M | 0.25M | 12.5ml(or1.864g) | | MnCl2 | 1M | 55mM | 5.5ml(or1.088g)addto100mlwithddH2O |
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