cDNA synthesis from total RNA

Chi-Kuang Wen cwen at aux.btny.purdue.edu
Tue Feb 6 19:48:59 EST 1996


CHISHOLM at BCRSSU.AGR.CA wrote:
>Has anyone had any success using oligo(dT) with total RNA to synthesize
>cDNA strands? I am trying to prepare a cDNA library from fruit tissue.
>I extract total RNA and then isolate the polyA+ fraction using an
>oligo dT column. The separation doesn't appear to be that good as indicated
>by the signals I see on a northern blot of the polyA+ fraction. My starting
>material (total RNA) gives very strong signals. Any suggestions are appreciated.
>Thanks
>J Chisholm


The oligo(dT) chromatography for poly(A)RNA enrichment works
very well for me in cDNA synthesis.  To prepare the ologo(dT) 
column, you need to re-htdrate the oligo(dT)-cellulose fully, 
and it would be better to remove the "fines" to prevent the
column from clogging.  However, I switch to using Promega
PolyATract and get successiful cDNA synthesis without using a
cDNA synthesis kit. The advantage of using the PolyATract is 
fast, and poly(A)RNA is resuspended in a small vol of water, 
and cDNA may be synthesized in a day starting from total RNA.

Regards,

Chi-Kuang





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