In article <Patterson.1122951948A at hsdndev.harvard.edu>, Patterson at cvlab.harvard.edu (Cam Patterson) writes:
>> 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
As you probably know, TAQ DNA Polymerase introduces an A into the
5' end of the PCR fragment. There are several commercially available
vectors (for instance, pGEM-T from PROMEGA) that use this peculiarity
to directly clone the PCR fragment.
Hope this suggestion could help you.
I am sorry you could not pass to the next round of the Soccer
virlab at dwarf1.quimica.uniovi.es