Help on PCR cloning

Ramanujam Raman ramjam at scripps.edu
Mon Dec 9 21:25:01 EST 1996


Dear Netters,

I am trying to clone a piece of promoter region using Linker PCR.  It is a
700 bp fragment from a larger piece in BSKS.  I made PCR primers that
would just give me this 700 bp fragment.  Both primers have linkers that
includes the four base in the sequence GGCC followed by a restriction site
sequence and then the actual primer sequence.  The forward primer includes
a HInd III site and the reverse primer includes a Bam HI site.  

The PCR reaction gave the product of the correct size with very litte
primer dimers.  I have phenol-Chloroform extracted the PCR product and
ethanol precipitated in the presence of glycogen.  I then used a fraction
of this product for restriction digest with Hind III and Bam HI followed
by ligation into the correspondingly digested BS vector.  Ligation was
carried out at 16 C overnight and then transformation using Xl-1 blue
cells from stragene.  

So far I get no colonies or only very few colonies that happens to be WT. 
I use insert to vector ratio of 4 or 5:1.  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?  Is it okay to clean the PCR
product by Phenol-CHCl3?  Is it okay to use glycogen to precipiate the
product?  Given the fact that there are 4 bases beyond the restriction
site, does either Hind III or Bam HI inefficient in digesting at ends?  I
would appreciate any help with respect to the above.  Please email your
replies to my email address.  Thanking you in advance,

RAM.



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