Cloning small PCR products

lmjakt at hkucc.hku.hk lmjakt at hkucc.hku.hk
Mon Feb 13 03:28:38 EST 1995


In article <asch-0602951745370001 at mac102219.med.cornell.edu>, asch at mail.med.cornell.edu (Adam S. Asch) writes:
> Does anybody have any suggestions for increasing the efficiency of
> subcloning short (100-200bp) PCR fragments into a vector such as TA? 
> Thanks.
> 
> Adam Asch, M.D.
> asasch at mail.med.cornell.edu
> 
> -- 
> asasch at mail.med.cornell.edu (Adam S. Asch)

I subcloned a fragment of about 200 bp straight into pKS, with no problem.
If the fragment is small enough, then it is likely that it will not contain
one of the two blunt cloning sites provided in pKS (dont know about other 
vectors, but as long as you have couple of blunt sites, should be OK), and 
you can use an in situ preparation of the vector to clone your fragment. Just
add your vector, PCR product, ligase, and appropriate restriction enzyme into 
the same tube. If your primers are not phosphorylated, then you can use excess
PCR product (I used about 30-50 times molar excess), the presence of the 
restriction enzyme lowers the enzyme, and I guess that you could add excess 
enzyme after ligation, to further reduce the background. Dont really know how
long you have to leave the reaction for (I have only done once, and had no 
problems), but I guess it depends on the amount of DNA and the relative 
amounts of enzyme activity you use. I used about 20 ng of vector, and left
o/n and o/d with a "recharging" of the enzymes after 12 hours. Gave me 60%
recombinant. Note that this is basically the cloning system supplied by 
Stratagene (I think!), except that the vector contains an eightcutting 
blunt site. (nsf I or  something like that). 
I like the method as it is extremely simple and doesnt require separate 
preparation of vector, or phosphorylation of primers or PCR products.

Martin

Dept. of Biochemistry, University of Hong Kong




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