From owner-biotechniques@net.bio.net Wed Apr 08 23:00:00 1998 Path: biosci!biosci!not-for-mail From: Thomas E Darga Newsgroups: bionet.journals.letters.biotechniques Subject: Blurry sequencing Date: 9 Apr 1998 07:36:01 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Approved: mmccarthy@BioTechniques.com Distribution: world Message-ID: <6gimch$17e@net.bio.net> Reply-To: darga@umich.edu NNTP-Posting-Host: net.bio.net Can anyone help me alleviate a blurry sequence problem with cDNA sequencing? I'm sequencing a randomly and oligo dt primed directional cDNA library and am having problems with the 3' end of the poly A containing clones. Also having problems with the clones containing poly nt stretches. The problem is that the sequence is clean from either end until I reach the poly nt stretch, when all lanes have bands of different intensities as if there are multiple cDNA species of a few bp difference in length. The mfgrs recommended reducing the extension temp of the PCR and Seq rxns, but it hasn't seemed to alleviate the problem. Thanks .