TA cloning...why is there no plasmid for BLUE colonies? - (May/26/2006 )
I ligated my ~181bp PCR sample with TA cloning kit....plate ligation samples onto X-gal/IPTG LB-Amp plate. i got LOTS of White but after double digestion, I found them not to contain my insert. I want to run the gel with a negative control so I can just do a 1RE cut to see whether I have insert or not. I tried to extract plasmid from BLUE colonies....Mini Prep seems to work okay ...but when I load the samples into my gel I see no bands for my negative control....
Does anyone know why? It grow in liquid culture (LB+ Amp)...just there are no plasmids....
hello, Getting no band for your negative means that problem lies in your plasmid do check it, or might be with ur T4 Ligase; use fresh. Secondly regarding Double digestion sometimes RNase treatment acts as a barrier and you didnt digestion but the plasmid appears as like that in estimation. i suggest u better go for digestion without RNase treatment and see what happens.
Thx^^ I will give it a try