Problem with cDNA synthesis

David A. Ray y4dar at ttacs.ttu.edu
Tue Jul 28 21:37:06 EST 1998


Hello all,

I just subscribed to this group so excuse me if this topic has already
been covered.

I'm trying to make a cDNA library from catfish kidney using Stratagene's
Zap cDNA synthesis kit, however, there has been serious trouble.  I can
get good mRNA (checked by denaturing gel electrophoresis and by reverse
transcription with both oligo dT and random primers then PCR to amplify
a known product).  I am consistently able to get cDNA using stock
reagents in the lab (MMLV-RT, 5x buffer, 10mM dNTP etc...) but, whenever
I switch to the kit reagents I fail miserably.  After doing first and
second strand synthesis, I fail to get any of the good PCR product that
I get when using stock reagents. The two major differences between the
Stratagene protocol and the protocol that works are the dNTP's and the
primer for 1st strand synthesis.  In the Stratagene protocol I am
required to use methylated dNTP's (5-methyl-CTP) and a linker-primer (GA
repeat+restriction site+TTTTT...).  I have tried various permutations of
both procedures with mixed results.

I don't want to give you to much information as I think it might bias
your responses.

Are there any thoughts on the problem?

Thanks for your help,

David

Texas Tech University
Lubbock, TX




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