zxmkr08 at mailserv.zdv.uni-tuebingen.de
Sun Oct 11 12:35:49 EST 1992
In <9210090425.AA07044 at cscgpo> Klaus.Matthaei at anu.edu.au writes:
>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
2. Preparation of polyA-RNA by using of oligo-dT columns.
3. Reverse translating of the RNA using the MMLV SuperScript from BRL and
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) */
/* krasel at chemie.uni-tuebingen.dbp.de (WIN/X400) */
/* "People are DNA's way of making more DNA." (anonymous) */
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