Help! PCR products cloning

Anon AL18 at le.ac.uk
Fri Sep 6 09:06:13 EST 1996


gene at PUBLIC.CD.SC.CN (Feng Lin) wrote:

>I am cloning DNA fragments amplified by PCR with Taq DNA polymerase.There are BamHI 
>sites at each 5'end of my primers and three additional bases(GTG or GCC) out of the 
>restriction site as protection bases.The experiments seem that the PCR products cannot 
>be digested by incubation with BamHI and cloned in pBS/BamHI.Other enzymes sites at PCR 
>primer(eg,PstI)can work well in my lab.The quality of primers may be O.K.But where's the 
>problem?
>Thank you for your help!
>
>Feng Lin

If you think you are having problems digesting at sites close to the ends of PCR fragments you could 
try concatermerising the DNAs into longer multimeric pieces.

Clean up your PCRs and then self ligate the fragments together using Klenow, T4 Kinase and T4 DNA 
ligase enzymes in a single reaction mixture - all these enzymes work effectively in 1x ligase buffer, 
under standard ligation conditions.  The result is long DNA concatermers (most BamHI sites are now 
internal and not on the DNA ends) which should be able to be efficiently digested.

I hope this helps.

Alan Lau

al18 at le.ac.uk
Dept. Microbiology & Immunology
University of Leicester
UK.  LE1 9HN.





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