Cheapest PCR product cloning technique

Todd Richmond todd at andrew2.stanford.edu
Fri Feb 19 13:46:20 EST 1999


In article <7opiGDf98QgB-pn2-94jVVSol8DWz at rnaworld.bio.ukans.edu>,
PGegen at UKans.nolospamare.edu wrote:

>On Fri, 19 Feb 1999 00:54:49, vrdefili at UCI.EDU (Victor DeFilippis) wrote:
>
>> Hello,
>> 
>> I'm looking for any and all input on the following issue.  I am considering
>> cloning PCR products from *A LOT* of specimens.  The fragments are between
>> 450 bp and 2 kb.  Along these lines I need to identify a technique that is
>> not prohibitively expensive, time consuming, or technically difficult.  I
>> would appreciate feedback from anyone who has an opinion on this and/or any
>> references.
>
>The simplest will be to use a proof-reading polymerase, which generates
blunt-end 
>products. Use primers which are not 5'-phosphorylated (to prevent
concatenation of 
>the PCR product). Clone the fragment directly into a blunt-end site of
the vector. 
>
I've had success using this method combined with pZero from Invitrogen.
Cut pZero for 30 min. with Ecl136 II, a blunt cutter that is easily
heat-inactivated. Throw in 5 ul of your PCR product with the vector,
ligate for an hour, and transform. The only colonies that appear have your
insert (well, most of the time). If I was doing a lot of these, I would
take 3-5 colonies from each transformation and PCR screen for inserts
using M13 forward and reverse.

Todd

-- 
Dr. Todd Richmond
Carnegie Institution of Washington
260 Panama Street                   Email: todd at andrew.stanford.edu
Stanford, CA 94305                 Homepage: http://cellwall.stanford.edu



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