5' RACE help

Colin Dolphin c.t.dolphin at qmw.ac.uk
Fri Apr 11 03:48:14 EST 1997


In article
<Pine.A41.3.95b.970409171659.88450A-100000 at homer33.u.washington.edu> N.
Salazar, nsalazar at U.WASHINGTON.EDU writes:
>Subject: 5' RACE help
>From: N. Salazar, nsalazar at U.WASHINGTON.EDU

>Hi there, I am trying to separate a short cDNA (50 bases) fragment from
>the primer (23 nt), after the first strand cDNA synthesis. This has been
>difficult since the primer and the cDNA are close in size and I need a
>clean cDNA for the c-tailing reaction.
>
>Any suggestions to get this cDNA clean from the primer are welcome. Recall
>also that the amount of cDNA that I obtain is very LOW and I have not been
>able to see it on acrylamide gels !!


Do you need to RT with a specific primer ? Using random hexamers works
well in 5¹RACE to prime synthesis of 1st strand cDNA and you can remove
them afterwards by passing the RT mix through a suitable column (eg.
Chromaspin -10). This step also removes the unused dNTPs which is VERY
important to do before the taling reaction otherwise you are unlikely to
get a homopolymeric 3¹ tail.

Colin



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