Hi,

I`ve been trying to isolate genomic library containing 160 kb BAC DNA using Nucleobond Xtra Midi, Vivogene BAC kit, Jetstar and a column-free method using Qiagene`s P1, P2 and P3 buffers. All kits were tested on a smaller high copy plasmid and they work.

Before cultivation of bacteria for BAC isolation, I plated them on agar plate containing chloramphenicol, IPTG and X-Gal. Took white colonies for cultivation. Colonies were small, around 1 mm in diameter.

I manage to isolate DNA but when I try to synthesise probes for FISH, either there is no probe smear on the gel, or there is smear but hybridisation does not work. Using chloramphenicol primers, I get a weak PCR amplicon and a lot of unspecific amplification.

Based on this I am asuming that i am mostly isolating "contaminating" bacterial DNA. Working with the same BACs about two years ago, there was no problem.

Any thoughts on what is going on with the BACs/bacteria?

Thank you,

Jana