RT-PCR

[Cornelius Krasel]

In <9210090425.AA07044 at cscgpo> Klaus.Matthaei at anu.edu.au writes:

>G'Day Netters

>I have some questions regarding PCR from RNA.  The standard method is to
>use Reverse Transcriptase and oligi-DT to synthesise cDNA from total RNA
>followed by standard PCR.  Another method is to use the dowmnstream primer
>for the cDNA synthesis followed by PCR.  More recently Amersham has brought

About a year ago, I worked as an undergrad student on a similar problem.
What worked fine for me, was:

1. Purification of total RNA by Guanidinium-SCN method (as described in
   Maniatis).
2. Preparation of polyA-RNA by using of oligo-dT columns.
3. Reverse translating of the RNA using the MMLV SuperScript from BRL and
   random hexamers.
4. Purifying the cDNA (with a Pharmacia Spun column, which is basically
   Sephacryl 2000, I think).
5. PCR using ordinary Taq polymerase.

We tried also PCR from cDNA prepared as above and transcribed with oligo dT
primers but got worse results.

Hope that helps, Cornelius.
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel at mailserv.zdv.uni-tuebingen.de (Internet)                  */
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/* "People are DNA's way of making more DNA." (anonymous)                  */