pET15b ligation probs.
Harry.Witchel at bristol.ac.uk
Sun Aug 6 05:06:06 EST 1995
In article <3vqqrr$f1b at lastactionhero.rs.itd.umich.edu>, jpsmith at umich.edu
(James Smith) says:
>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
>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.
>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
>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
>were observed (transformation was successful on the pUC18 control
>I'm at a loss. I've been trying to get this to work for about a month
>with no success. I'm new at these procedures, but it doesn't seem like
>should be this problematic! The smear on the gel described above
>me - it seems that if my insert was dimerizing, etc., I would see
>bands corresponding to 1.5kb, 3kb, 4.5kb, etc. instead of a smear.
>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
>University of Michigan
Be brave! You are not alone. Getting a "trivial" PCR product
ligation to work has wasted many lab-months. It looks like you are
receiving quite a few protocols for doing this, which is nice. The thing
to remember is that there are hundreds of ways of doing a ligation
correctly, but there are millions of ways of doing it incorrectly; every
single technique ever published works -- in at least one laboratory.
Obviously getting a technique to work in your laboratory is a matter of
remembering to do all the steps "between the lines" in whatever protocol
One control experiment you did not mention is to take your BamHI
CIAP treated vector, kinase it, and autoligate it side by side with your
other ligations and then transform; this is a much more useful control
than a pUC18 transformation because it tells you: your bugs are competent
and you are using enough vector (and that the vector is not contaminated
with a ligation inhibitor). As usual with the potitive control, the
negative control is to transform with the linear CIAP vector (which should
still offer no colonies).
All that being said, I think it likely that the kinase control
will work for you (ie your vector is OK), and that the problem is that
your PCR product is dirty, as PCR products typically have all sorts of
junk with them that make them hard to clone. At least you will be certain
that the problem is cleaning up your PCR product (rather than bad vector,
bugs, or ligase). A confidence builder is to use your vector to clone any
old insert from another vector you have lying around: it doesn't solve
your problem, but at least you cloned something.
In terms of cleaning up PCR products for cloning, the method of
choice is running a NuSieve LMP agarose gel (no financial connection to
FMC) and then cutting the band out of the gel and getting the DNA out of
the agarose. There is a huge thread in this newsgroup concerning the 1000
ways of getting DNA out of agarose (and of the 1,000,000 ways of not
getting it out of agarose).
Courage. Remember that the longbowmen at Agincourt defeated the
French knights who had armour and were on horseback, because the English
University of Bristol
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