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

[Annette C. Hollmann]

>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					   _/\_

I had the same problem.
The conditions I was using were:
1 ug RNA (not DNAse treated)
dNTP's and all other reagents as per instructions included with the kit.
I used the random hexamer primers.

Before any reagents were added, the RNA was heated to 65C, 5 min, quenched
on ice.
After adding the reagents , I left the samples at room temp for 10 min.
The cDNA synthesis was at 42 C, 15 min.
I also did not get any product.

I made the following changes:
1. instead of incubating at room temp for 10 minutes (room temp was
sometimes as low as 16 C) I used 25 C.
2. I increased the cDNA synthesis to 60 min.
These conditions repropducibly resulted in cDNA synthesis

If this does not work for you, make sure you inactivated the DNAse.

I hope this helps
Annette
ah690549 at mbcr.bcm.tmc.edu

N.B. According to Perkin-Elmer techies, the 10 minute incubation at 25 C
prior to cDNA synthesis is essential for proper annealing of the random
hexamer primers