Subcloning PCR

eb86336 at inet.d48.lilly.com eb86336 at inet.d48.lilly.com
Mon Sep 12 19:06:15 EST 1994


We have had a lot of trouble using the commercially-available "TA" cloning
vectors.  They work fine just after you buy them, but as they sit in the
freezer, they start to lose their effectiveness, and the number of
transformants quickly begins to drop.  Stratagene has a new kit called
pCR-Script & pCR-Script-cam, that utilizes an 8bp restriction site, SrfI, that
they have placed in their multiple cloning site.  A population of PCR products
has no 3' A extensions, and are blunt.  This subpopulation can be cloned into
the blunt SrfI site.  To facilitate a greater pecentage of isolates with
inserts, both SrfI and T4 ligase are added to the ligation reaction. 
Therefore, if any plasmid self ligates, SrfI will cut the plasmid so that an
insert can be ligated in.  The only caveat is that you cannot have a SrfI site
in your gene or regenerate a SrfI site with the 5' end of your PCR primers. 
This is not that difficult to do as the site sequence is GCCC'GGGC, and this
sequence rarely shows up in most genes (unless you are cloning a gene from a
high %GC organism like Streptomyces sp.).  This kit works for us ever time, we
usually get 90-100% of white colonies being the correct recombinant.  The
pCR-Script-cam system uses a CAT gene instead of an Amp gene, so that if you
were PCRing from an Amp-r plasmid template, you do not have to worry about
screening false positive Amp-r colonies.

Mike Menke
Eli Lilly and Company



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