Help on PCR cloning

Vladimir Svetlov vvsvetlov at utmem1.utmem.edu
Tue Dec 10 08:58:02 EST 1996


In article <ramjam-0912961825010001 at 137.131.48.69>, ramjam at scripps.edu
(Ramanujam Raman) wrote:
>  Has anybody out there tried PCR
> cloning successfully and if so, does any of the above step seems odd or
> inhibitory in any way to the cloning process?  

Glycogen in the precipitation mix definitely looks odd.

> Is it okay to clean the PCR
> product by Phenol-CHCl3?  

Yes.

>Is it okay to use glycogen to precipiate the
> product?  

Don't know but WHY? One good thing about PCR is that you get a lot of
product and you don't need to add anything but salt and ethanol to
precipitate it. Secondly, polysaccharides are often inhibitory for cloning
enzymes. Loose the glycogen. If you get so little of a product that
addition of the carrier is inevitable use tRNA instead. 

> Given the fact that there are 4 bases beyond the restriction
> site, does either Hind III or Bam HI inefficient in digesting at ends? 

First you need to make sure that your oligos contain those 4 bases.
Digestability of the PCR product is easy to check by kinasing it with
P32-gammaATP, digesting with appropriate enzyme and running the mix
through G-25 column to see if the label remains on the column. HindIII
might be a suspect if your oligo is 1 or 2 bases short, since its not very
active on the ends. You can consult NEB catalog for overhang size effect
on digest activity. Again its easy to check using kinasing experiment
above. 
Remember, that using BamHI you need to PCI (or deproteinate in any other
way) your digests because BamHI is not inactivated at 65oC. Presense of it
in the ligation mix would definitely antagonize the ligase <G>.
I usually purify PCR from the gel, extract twice with PCI, precipitate
twice before digest. Vector always SAPed to deminish the background. 
Hope this helps.
Regards,
Vlad



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