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
advance.  
*******************************************************************
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
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________



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