joining two fragments with PCR

Richard V. Giles giles at liv.ac.uk
Thu Oct 1 07:48:09 EST 1998



Andrew Schrader wrote:

> I have generated two fragments from a cDNA template using PCR. I would like to
> join these two fragments in a PCR reaction to amplify a length of DNA that is
> shorter than the original cDNA (70 bp - to be used in competitive PCR).
>
> One fragment is 210 bp and has a 30 bp sequence at the 3'-end, homologous to the
> 5'-end of the larger 580 bp fragment. Two primers will amplify up the whole
> length of the two fragments if they anneal to each other after strand
> denaturation.
>
> So far however, I have only managed to see a smear on my gels that stretches
> from my primers to about 1500 bp. A positive control template that uses the same
> primers, amplifies up fine.
>
> Any clues?
>
> Andrew

What I did in a similar situation was to perform 2 quasi-exponential PCRs for the
initial products such that a preponderance of the stands which cross hybridize at
their 3' ends were formed. Cleaned up the DNA (phenol ex, etOH ppt) resuspended the
products in water. Set up PCR reaction with small quanitity of both initial reaction
products. I then performed one round of PCR without added primers and using a high
(~70 C) annealing temperature. I then performed a low cycle number exponential PCR
(15 or so cycles) with the primers, using a Ta appropriate for the primers.

Remember that if you over-cycle with a Ta below ~65C then "semi - random"
multimerization of the PCR products can occur - reference in Nucl Acids Res some
years ago I think- producing nasty smears as you describe.

Good luck


--
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
http://www.liv.ac.uk/~giles/





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