5 prime Race-where do you start?

Eric Lader lader at ambion.com
Wed Oct 9 20:07:08 EST 1996


In article <53h25p$1rp at is.bbsrc.ac.uk> Clive Tregaskes,
Clive.tregaskes at bbsrc.ac.uk writes:
>1 two start points (tho' Northern suggests this isn't so)
>2 short transcripts are due to secondary structure stopping the RT
>3 long transcript is due to DNA contamination of my RNA prep (but
>contains a dC tail)
>Lots of ideas but no answers, so does anybody know how you tell the
>real start site from any secondary structure artifacts.

Design a primer to the 5' end of the new sequence and do standard rtPCR
to see if you get the predicted fragment.

Use the novel sequence as a probe on a northern blot and see if you pick
up the predicted message.

Use the new sequence on a genomic southern and see if it maps to the same
fragment as the rest of the sequence (need to maybe use a rare cutter)

Hope this helps.

               **my opinions are just that**
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        Eric Lader Ph.D.	Senior R&D Scientist,  
                          Ambion Inc.
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