KlentaqLA extra A

Wayne M. Barnes, Ph.D. barnes at BIODEC.WUSTL.EDU
Fri Nov 15 17:35:19 EST 1996


554 <craig at primer.yale.edu>... 550 Host unknown (Authoritative answer from name server)
Subject: Re: Klentaq LA

Dear User of KlentaqLA ("Advantage Klentaq" from Clontech)

    You ask:  Does KTLA put the extra A onto PCR products?

    Klentaq1 alone does put the extra A on, so the question is, is the
proof-reading enzyme keeping it cleaned off?  

    I haven't done the experiment, but when I do, I will carefully test
several post-PCR treatments:

     1 - Add EDTA to the PCR reactions while they are still hot.

     2 - Let the PCR reactions sit on my bench while I eat lunch.

     3 - Let the PCR reactions sit in the refrigerator overnight.

     1B,2B,3B - All of the above with or without an extra 10' on the final
extension step.

   I expect that the amount of extra A will vary under the above
treatments.  Since Klentaq1 survives phenol and ethanol, and some dATP
does, too, it could even be that Klentaq1 puts the extra A on during the
a 37 deg. ligation (but much less during a 15 deg. ligation).  This would
mess up both the target and the vector.  

   In fact, I always get plenty of clones by ligating into a blunt vector.
I kinase my PCR primers so that the PCR product has phosphates, and I
treat my vector DNA with calf intestine alkaline phosphatase.  [ I cut 
my vector with BamH1, then C.I.P., then fill in with Klenow + 4 dNTPs.]
I always include 1 mM hexamine cobalt chloride in my blunt ligations.  

   Several people have reported to me that they get plenty of clones of
KTLA PCR products into T-vectors.

   So -- you can have it both ways.  There are at least some ligatable
blunt ends, and some with extra A, but it may depend on exactly how you
bring down your PCR reactions.  


On Thu, 14 Nov 1996 14:19:03 -0800, 
Craig Cinquina   <craig at primer.yale.edu> wrote:

>Dear Dr. Barnes
>I have been using Klentaq-LA after reading about it in your 1993 paper. 
>I was wondering if you knew whether Klentaq-LA would leave an A on the
>ends of the PCR product (like Klentaq alone would) or if the PCR ends
>would be blunt ended due to the PFU. Please let me know at your earliest
>convenience. 
>Thank you 
>Craig Cinquina
>craig at primer.yale.edu
>


Wayne M. Barnes, Ph.D.             fax: 314/362-7183  or 362-3350
Dept. Biochemistry 8231, Washington Univ. Med.School
4566 Scott Avenue   Barnes at biodec.wustl.edu http://mbb.wustl.edu/~barnes/
St. Louis, Missouri 63110 USA       ph: 314/362-3351



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