Help - Cloning Problems
hroychow at NMSU.EDU
Tue Jun 20 15:57:03 EST 1995
On 20 Jun 1995, James Smith wrote:
> Hello, all.
> I am attempting to insert a DNA fragment into a pET-15b plasmid vector. I'm
> running into problems.
> The DNA fragment has XhoI restriction sites on both ends, and is approximately
> 1.5kb in length.
> I tried digesting both the plasmid and DNA fragment (separately) with XhoI
> (3 hrs @ 37 deg. C; the plasmid was checked for complete digestion by
> running on an agarose gel) and then treating the plasmid with alkaline
> After an attempted ligation with T4 DNA ligase (16 deg. incubation overnight),
> I ran the ligation mixture on a gel and it appears that the plasmid did not
> take up the DNA, and also did not recircularize.
> If a plasmid has been treated with alkaline phosphatase, would it still
> recircularize during ligation even if it does not take up the insert?
Dephosphorylated vector should not recircularize by itself. So, your
> Should I be treating my DNA insert with alkaline phosphatase as well since
> both ends contain the XhoI restriction site? If both DNA *and* plasmid
> are treated with alkaline phosphatase, will ligation still occur?
The insert should not be CIP'd. Since you are doing sticky, identical end
ligation, vector dephosphorylation is recommended strongly. Both the
molecules should not be CIP'd; no ligation will occur if they are.
> (Ligation mixture includes Tris-Cl, DTT, ATP, DNA insert, plasmid,
Try using PEG8000 at a final conc. of 5% in your ligation mix. How many
times have you tried doing the ligation? First timers often have
difficulty, and you may get it work in another couple of tries. If you do
not (with the same preparations) then I would venture a guess that you
have a problem of the phosphatase being carried over into the ligation
mix. Try going through an earlier thread on the net about cleaning up the
vector DNA following CIP'ing. If you need, I will fwd you the protocol.
> Any help would be greatly appreciated. Please email me if you would like
> more information.
> Jim Smith
> jpsmith at umich.edu
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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