Help - Cloning Problems

James Smith jpsmith at umich.edu
Tue Jun 20 11:02:45 EST 1995


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
phosphatase.

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?

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?

(Ligation mixture includes Tris-Cl, DTT, ATP, DNA insert, plasmid, H20.)

Any help would be greatly appreciated.  Please email me if you would like
more information.

Thanks,
Jim Smith
jpsmith at umich.edu




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