PCR amplification of cloned insert

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Mar 23 12:53:18 EST 1995

KA Gilbert (kgilbert at cmp.hmc.psu.edu) wrote:

| I have a 2kb insert cloned into Bluescript and access to a
| thermocycler. I would like to PCR amplify this insert, but have no PCR
| experience.  The vector has T3 and T7 promoters and insert is arranged
| with T7 transcription for antisense.  No sequence data is available. 
| Could someone please inform me of reaction conditions and cycle
| profiles to PCR amplify this insert.

A simple solution is to buy an ABI PCR Module that contains everything you
need - pUC119 control DNA with 678 bp insert, forward and reverse primers
which anneal within the lacZ' region, buffer, and Taq polymerase. Follow the
directions and you should have no problem since the conditions (cycle
temperatures and MgCl2 concentration) have been optimized for these primers
and template. pBluescript also has the lacZ' region. One problem would be if
your insert has sequences which match the primers, but since you don't know the
insert sequence, you'll have to do it blind anyway.

* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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