making concatmers of PCR products

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri Aug 27 13:36:00 EST 1993

-------Morrie (boy, I am getting sick of PCR) Manolson writes--------------
Dear netters,  I have heard reports of people first 
making concatmers of PCR products
to improve the cutting of the PCR product with restriction enzymes
prior to ligating into the vector of choice.  I have also heard 
(or think that I have heard) that people are just throwing in 
some ligase after PCR (no fill-in, no nothing) and that this
is enough for forming concatmers.  Is this true?  I would appreciate
any and all protocols for making concatmers of PCR products.
I will of course post a summary at the end.  Thanks in
Hi Morrie
    I too have noticed this little trick on the net!  I think I will
try it the next time i need to clone a pcr product that has restriction sites
at the ends of the primers.  Ligation from a PCR reaction may work 
but don't forget to add ATP for the T4 DNA ligase.  You may need to add
a bit more MgCl2, say to 10 mM, to get the ligase to work properly.  The 
only worry is if Taq has put on that extra A (or is it T?) I'm not sure 
what proportion of the PRC product will be ligatable.  Can anyone out there
answer this question? 

Dan Gietz
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
"Trying to do the Manitoba Thing"

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