cDNA Challenge*************

[douglas l feinstein]

Subject: cDNA Challenge*************
From: Shahram Mori, smori at nmsu.edu
Date: 24 Sep 1994 20:55:30 GMT
In article <3623o2$l5t at dns1.NMSU.Edu> Shahram Mori, smori at nmsu.edu writes:
>
>Dear Molbionetters,
>I have been using the Perkin Elmer RNA PCR kit to make cDNA from my Total
>RNA samples. My RNA is in good condition and has been DNase treated. I
>follow the protocol in GeneAMP kit and yet achieve no cDNA's. My
>concentration of RNA is 200ng. As this is being done in a procaryotic
>system, I am using random hexamers for cDNA synthesis. The time of
>synthesis is 15 minutes at 42C. The dNTP concentration is 1mM. Anybody
>that has any recomendations as to how I can improve this reaction , I
>would be grateful to. 
>Thanks a milion,
>cheers
> 
>-- 
>Shahram Mori					   _/\_
>Program in Molecular Biology			  _\  /_
>Dept. of chemistry and Biochemistry Box 3C	  \_  _/
>NMSU  Las Cruces NM				    ||


Maybe increase the time to 1 hr. After 1 hr, add a second aliqout of RT,
and do another 1 hr. At end of reaction, heat 95oC for 5 min to kill RT.
We also use higher dNTPS...i think it is2.5 mM each
good luck

d.l. feinstein