pET15b ligation probs.

Ted M. tedm at darkwing.uoregon.edu
Thu Aug 3 21:53:47 EST 1995


In article <3vqqrr$f1b at lastactionhero.rs.itd.umich.edu>, jpsmith at umich.edu
(James Smith) wrote:

> 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. 

> 
> Jim Smith
> University of Michigan

Jim, I'm concerned about the "purification" of the BamH I digested insert,
it is essential that you get rid of the cut ends, primers and dNTP's to
get a good ligation. Good ways to purify this insert include isolation
from an agarose gel or a size exclusion method. Typically I'll use a
Microcon 30 (from Amicon) spin unit to remove all the cut ends and
dNTP's/primers from a PCR/digestion. This leaves you with a little
template DNA and your primary PCR product with digested ends. If the
amplification isn't very clean, a gel is the way to go. With all the ends
and/or primers present it is possible to get big ugly TA ligated smears
and the like. If the Prep-a-Gene kit is used in conjunction with gel
purification, ignore this point, as I am unfamiliar with this procedure.
   Concatamers are a problem with an insert with compatible ends. If this
is the case try a lower ratio of insert to vector and possibly PEG to help
your chances. Good luck.

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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