pET15b ligation probs.

[James Smith]

I have posted here before and have corresponded with some of you via
email, but I'm still having ligation problems.  I have recently switched
restriction sites in hopes that I may have better luck.  I have been 
successfully amplifying a 1.5kb DNA fragment via RT-PCR with a Bam HI 
restriction site included in the primers, 3 bases from the end of the
fragment (according to NEB's catalog, Bam HI appears to be a good end-cutter).
I've been digesting the plasmid with Bam for 3h (checked on gel for
complete linearization) and then treating with CIAP.  I've been using
Bio-Rad's Prep-a-Gene kit to purify the plasmid.  The DNA insert was
digested for 3 h with Bam and also purified with Prep-a-Gene. 

My ligation mix has included DNA & plasmid in a 3:1 (approx.) ratio.  I've
been using 2 U of T4 DNA ligase (BM) and the included 10x ligase buffer.
My ligase appears to be good as it ligated a HindIII lambda digest ladder
in an hour at RT.

Following an hour ligation, I ran 10 uL of the 30 uL ligation mix on a gel
and noted a fairly intense smear from my 1.5kb fragment up to a point 
corresponding approximately to the location of circular plasmid with no
insert (although no *distinct* bands were observed).  The smear continued
up beyond the location of linear plasmid, but was less intense in this 
region.  I attempted transformation with this ligation rxn, but no colonies
were observed (transformation was successful on the pUC18 control plates).

I'm at a loss.  I've been trying to get this to work for about a month now
with no success.  I'm new at these procedures, but it doesn't seem like it
should be this problematic!  The smear on the gel described above concerns
me - it seems that if my insert was dimerizing, etc., I would see distinct
bands corresponding to 1.5kb, 3kb, 4.5kb, etc. instead of a smear.  However
I've never seen a successful ligation rxn run on a gel.

I'd appreciate any comments or advice anyone could offer.  Thank you very
much!

Jim Smith
University of Michigan