TRANSFORMATION (ONE-STEP PEG METHOD) 1.Dilute 1:100 a fresh O/N culture of bacteria into prewarmed LB broth and incubate cells with shaking (225 rpm)to an OD600 of 0.3-0.4. 2.Add an equal volume of ice-cold 2X TSS and mix gently.[TSS is LB broth with 10% PEG (MW3350-8000),5% DMSO,and 20-50 mM Mg2+ (MgSO4 or MgCl2)at a final pH of 6.5]. 3.For long-term storage,cells are frozen immediately in a dry ice/ethanol bath and stored at -70℃. For transformation,a 0.1 ml aliquot of cells is pipetted into a cold polypropylene tube containing 1μl (100 pg)od plasmid DNA,and cell/DNA suspension is mixed gently.[When frozen cells are used,cells are thawed slowly on ice and used immediately.] 4.The cell/DNA mixture is incubated for 5-60 min at 4℃. 5.A 0.9 ml aliquot of TSS (or LB broth)plus 20 mM glucose is added,and cells are incubated at 37℃ with shaking (225 rpm)for 1 hour to allow expression of the antibiotic-resistance gene. 6.Transformants are selected by standard methods. PNAS (1989)86:2172-2175.CT Chung,SL Niemela,RH Miller. |
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