Help! PCR products cloning

Bernard Murray bernard at elsie.nci.nih.gov
Tue Sep 10 10:32:05 EST 1996


In article <323416A3.41EF at zoot.tau.ac.il>, gabiger at zoot.tau.ac.il says...
>
>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!

>It seems that BamH1 needs more then 3 bases at each side of the restriction 
>site in order to cut. There are some cataloges which that kind of 
>information is writen. Try to check it. If this is the case you will have to 
>design new primers with longer tails.

No, the table in the NEB catalogue claims BamHI cuts with 97% efficiency
when only 1 base from a HindIII cut and also shows that >90% of a BamHI ds
oligo with a 2 base extension at either end is cut in 2 h.
	We have found bad batches of BamHI that have a markedly reduced
cutting ability so maybe try another lot of enzyme.  Also, BamHI can
be a little fussy over the reaction conditions (although most of the time
the result is star activity rather than lack of activity).  Did you
purify the PCR product at all - maybe the pH is a little too high?
Any chance that you are forming really long tandem repeats during
the ligation that inhibit transformation? (the digestion will leave
phosphorylated ends).
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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