RT-PCR Question

Robin Beech robin_beech at maclan.mcgill.ca
Fri Oct 18 09:16:28 EST 1996


In article <e-anderson-1710960918220001 at news.ski.mskcc.org>,
e-anderson at ski.mskcc.org (eric anderson) wrote:

>(theoretically) have an internal standard.  alternatively, you could make
>a construct of your gene of interest with a deletion between your RT-PCR
>primer sites and use a known amount of that as a competitive target.  this
>is probably a more accurate internal standard than what we do, but since
>we're working with 30+ genes, the construct making is a bit time
>consuming.  (see Biotechniques 21:268-279 and 280-285(Aug. 1996)) for more
>info. on this.

One alternative technique to making a specific construct as an internal
control is to do a very low stringency PCR, cut out and clone a fragment a
little larger, or smaller than your target. The fragment will contain junk
probably but should have the correct primers on the end. There are obvious
problems with this approach in that the amplification kinetics may be
slightly different between the target and competitor but if the appropriate
tests are done it would save a lot of time for each construct.

Robin Beech
Institute of Parasitology
McGill University



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