Cloning a PCR product

Cam Patterson Patterson at cvlab.harvard.edu
Sat Jun 25 12:11:48 EST 1994


Friends,

I have a somewhat difficult (for me) cloning problem. 
I have a PCR product amplified with a 3' primer which
inserts a HinDIII site into the product. The 5' primer,
however, does not introduce a restriction site. I would
like to clone this directionally into a reporter vector
(pGL2). My vector's polylinker has a 3'HinDIII site and a 5'
Sma site. I'm hoping I can clone my PCR product into
the blunt-end Sma site and the HinDIII site and
my dilemma is what is the best way to do this (short of
designing a new 5' primer and introducing another restriction 
site)? These are the options I see:

1) Clean up the ragged PCR termini with Klenow and then
cut with HinDIII, then clean and clone into my cut vector
(and if I do this, can I just add Klenow to my PCR mixture
or do I have to clean up the product first?).

2) Simply force clone the 5' end into the Sma site, hoping
enough of my PCR product is blunt-ended to be clonable.

Does anyone have any experience with such a problem, or
any other ideas? Thanks.

Cam Patterson
patterson at cvlab.harvard.edu



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