polyA problem with ESTs

Mr.M.R.M.Botcherby mrbotche at hgmp.mrc.ac.uk
Thu Dec 20 09:34:32 EST 2001

Use 20-mers
With your usual sequencing conditions but with 3 times the amount 
of primers (as only one subset is going to work in each reaction,
each one of the TTT...C, TTT...G and TTT...A needs to be at the 
optimal concentration.

Good luck,


In article <9vsrrh$4pt$1 at mercury.hgmp.mrc.ac.uk>,
	Olin Anderson <oandersn at pw.usda.gov> writes:
> We have a large EST project and are having trouble with the
> the 3' sequencing - reading through the polyAs.  More than
> 30 A's and the sequence is short, longer than 50 and we get
> no good sequence.  I've heard some sites (but not all) have
> had success with the oligodT anchored primers.  Could anyone
> share the conditions/primers/etc they use to successfully get
> long reads past cDNA polyA stretches?  Thanks.
> Olin
> ---

Marc Botcherby
Mouse Sequencing Group leader UK-HGMP
m.botcherby at hgmp.mrc.ac.uk
Tel:01223 494555 Fax: 01223 494512

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