the problem on plasmid ligation and transformation

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Aug 17 03:30:54 EST 1999



"Dr. D. Zha" wrote:

> Hi,
>
> I have met an old problem, when I try to clone the PCR product which is
> cut by two different enzyme into a plasmid, it does not work. The length
> of the plasmid is around 6 Kb and the insert is around 1 kb, and recover
> the DNA bands after digestion from agarose gel by kit, and using T4 DNA
> ligase which bought from Boheringer, and the competent cell was prepared
> by the method of RbCL which I used the my former laboratory quite
> successful, and the transformation of the cell can reach 10 to 6 cfu/ug
> the plasmid, would please help me to solve the problem, I appreciate any
> helpful suggestion, Thanks!
>
> Zha

Hi,
There are two things in your post to comment on:
1. Cloning PCR products has often been considered difficult. If you use Taq
polymerase for amplification, a TA vector might be helpful for cloning
(However, Taq is definitely not a good choice when you want to clone the
product because of  the high error rate of the polymerase! Well of course
it depends on what you want to do with cloned fragment. If it is only to be
used e.g. as a hybridization probe it is ok to use Taq, but in case you
either want to sequence the product or use it to make recombinant proteins,
I strongly recommend using a proofreading polymerase). As to your problem
of cloning I suggest ligating the PCR products to a concatemer to avoid
problems associated with RE digests close to DNA ends. I usually purify the
PCR product, take it up in a small volume (10ul), add ligase buffer, T4
polynucleotide kinase (1unit; needed to phosphorylate the 5´ ends of the
product) and ligase (1unit), incubate for 4h at RT. Ligase is then
inactivated by incubation at 70degrees for 20min. It is usually a good idea
to take a small aliquot at this stage to control the efficiency of ligation
(sample 1). Then, perform your RE digest as normal, and take an aliqout
afterwards (sample 2). Run both samples on an agarose gel. A ladder of
concatemers should be evident in sample 1, and only momomeric fragments
again in sample 2. If the control gel is ok, inactivate the enzymes (by
phenol/chlorofom preferably), and ligate to vector as you normally would
do.
2. you say the efficiency of your cells "can reach 10 to 6 cfu/ug the
plasmid". This is NOT a good efficiency at all. For simple subclonings it
might be sufficient, but in case your cloning is slightly more complicated,
it is way to low. What you should have is at least 10 to the 8 cfu/ug, the
more the better. You might consider making new cells with a different
method (Inoue´s protocol is considered the best in the moment, you´ll find
it in the FAQ list of this newsgroup), perform electroporation (if an
electroporator is available to you), or buy chemically competent bacteria
from companies such as stratagene (no affiliation). They are not too
expensive as long as you take them for difficult clonings only....

Hope this helps,
Frank




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