2.Growin250ml"SOB"at18CuntilOD600=0.6. 2.Onicefor10minutes. 3.Spinat2500xg(5000rpminaSorvallGSAor3000rpminaBeckmanJ-6Bcentrifuge)for10min.at4C. 4.ResUSPendcellsgentlyin80mloficecold"TB".5.Onicefor10minutes. 6.Spinat2500xg(5000rpminaSorvallGSA,5500rpminaSorvallSS-34,or3000rpminaBeckmanJ-6Bcentrifuge)for10min.at4C. 7.Resuspendcellsgentlyin20mloficecold"TB". 8.AddDMSOtoafinalconcentrationof7%. 9.Placeonicefor10minutes. 10.Aliquotinto1-2mlandfreezeinliquidnitrogen. 11.Storeinliquidnitrogen.Procedure
1.InoculatefromanovernightgrowninLB.SOBMediumandTB(TransformationBuffer)
SOB 2%(w/v)bactotryptone TB 0.5%(w/v)yeastextract 10mMPipes 10mMNaCl 55mMMnCl2 2.5mMKCl 15mMCaCl2 10mMMgCl2 250mMKCl 10mMMgSO4 adjustpHto6.7with5NKOHpriortoaddingtheMnCl2thankstoMarkusSchneemannforthetip! pH6.7-7.0