In article <9501160241.AA94256 at lamar.ColoState.EDU>,
xuhu at LAMAR.COLOSTATE.EDU (XuHu) wrote:
> Dear Netreader:
> We are having a great difficulty in cloning lambda genomic fragments(
> one 4.5 kb and one 14 kb) into pBluescript xho 1 site. We grew the white
> transformants and checked inserts by restriction digest. We found all of
> them (from both fragments cloning) are deleted pBS vector (about 2.2 kb)
> without inserts. Check ligation there was shiftup on gel compared with
> self-ligated pBS. The gene we are cloning may involve in cell division of a
> plant. Can anyone give some suggestion or comments.
> Xu Hu
Are you concluding that the vector is deleted because it no longer cuts
with XhoI and looks smaller on a gel? Are you sure it cut at all? Do the
bad clones cut with anything? Are you doing alkaline minipreps? I had a
problem like this for a while which went away (and showed that all my
cloning was still working) when I switched to alk preps using 0.15N NaOH
instead of 0.2N.
I have also tried to clone genes that appear to have a lethal effect on
bacteria: my other suggestion is to plate the putative transformants on
plates with IPTG. I assume (with no proof) that running a lot of
transcript off the lac promoter could overcome any transcripts being read
off "internal" promoters at least in one orientation by being antisense.
Oh well, it worked for me. Good luck if neither of these suggestions help.
cas9 at cornell.edu