Help on PCR cloning

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue Dec 10 11:14:02 EST 1996


At 06:25 PM 12/9/96 -0800, Ramanujam Raman wrote:
>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.  
>
>snip<
>
>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.
>
>

Cloning of PCR products is a routine thing in our lab. I personally use either of the two methods: 1. pGEMT vector or 2. Blunted vector. 

If you do not want to use TA cloning strategy, upon completion of the PCR, simply polish the ends of the products by incubating the PCR reaction mix with *extra dNTPs* (about a 5-10pmol) and an unit of Klenow for 15 min at 37 C. Then run the entire reaction on agarose gel (at a preparative scale), recover the PCR product(s) from it (using any of the number of methods available) and into dH2O or TE8. Use that in a ligation reaction involving a blunt-cut vector (eg. SmaI-digested pGEM3, pUC18/19 etc.). You can recover your insert after that using the built in sites. 

The pitfall in not obtaining the pCR products from a gel is that there will always be a molar excess of the primers left over from the reaction and there would be the so-called 'primer-dimers' that would interfere with ligation.

Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu



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