New England Biolabs
NEB 5-alpha High Efficiency Competent E.coli Cells
C2987I
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I have used NEB 5-alpha high efficiency competent E. coli cells for transformation of expression plasmid of size 6.3 Kb by heat shock method. After transformation, transformed cells were recovered and streaked onto selective agar plate. Got around 3000 colonies per 200 ng plasmid construct being transformed. 8 out 10 colonies screened by colony PCR gave correct clones.
Transformation of expression plasmid constructs for protein expression
200 ng plasmid construct per 50 microliter NEB 5-alpha high efficiency competent E.coli Cells
Transformation protocol which I followed was long incubation protocol and heat shock method, which comes with instruction manual with these competent cells. Simply thaw the cells on ice for 20 minutes, add 200 ng of your plasmid construct and incubate on ice for another 30 minutes. Then heat shock the cells with plasmid at 42 degree C in a water both 45 seconds. Then place the heat shocked cells on ice for 5 minutes. Then add 950 microliter of pre-warmed SOC medium. Recover at 37 degree C for 90 minutes with shaking. Plate neat and several dilutions of transformation mix on to selective agar plates.
Thaw cells completely. Always perform positive control transformations (pUC19 DNA which comes with the kit).
Satisfactory transformation of my expression plasmid construct into E. coli cells. Got 3000 colonies per 200 ng plasmid construct being transformed by heat shock method.
N/A
Simple and easy protocol. Kit is not expensive compared to other providers.
None
Overall, very satisfied with using NEB 5-alpha high efficiency competent E.coli cells for my routine expression plasmid constructs.