pICZa transformation problem - (Nov/01/2007 )
I have inserted a 1.5KB gene into pICZa vector and prepare to transform into XL-1 Blue chemically competent cell.
Im pretty sure the ligation is successful because I checked on the agarose gel there are fading and shift of bands.
However, after transformation and 24 hours incubation on 25ug/ml zeocin LB agar, no colonies grow on it.
I did a positive control where the pICZa was not cut by the restriction enzymes(EcoR1, Not1), there are numerous colonies grown after transformation.
I even tried electroporation, failed.
What's my problem?
Actually I think my ligation should be ok.
But I double digest my vector and genes using EcoR1 and Not1 from NEB, using NEB EcoR1 buffer as they recommended.
Do I need to do sequencial digest?