Sequencing 3'RACE PCR products: help on dTVN primer?

Stan Stan
Thu Aug 14 16:51:45 EST 1997


Having a nice summer everyone?

My group is doing high-throughput (400-800 PCR products per day)
sequencing of 3'RACE PCR products with TaqFS and dye terminators.  The
products are easily sequenced from the 5' end with a standard primer.  I
want to obtain sequence from the 3' end as well.   The RT-PCR products
have very long stretches of polyAs that cannot be read through using the
3' end primer.  Can one use an anchored polyT primer such as dTVN, where
V= A,G, or C?  I have tried this, but have had no success - multiple
priming, just plain messy data.  Does anyone have any suggestions as to
what can be done??  Reaction conditions, etc.???

Sincerely Stuck at the 3' End,

Stan



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