cDNA synthesis from total RNA
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.
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.
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