problems with sequencing in big dye v3

eve evetoulza at free.fr
Sat Oct 12 06:00:05 EST 2002


Hello everyone

We are generating ESTs libraries cloned in pCRII-TOPO plasmid (TA cloning).
Inserts are generally about 200-1000 bp with our method (ORESTES). Since
several weeks (new version of the big dye kit = v3), we have got lots of
sequencing problems, quality of the sequence is very poor (10-30% nice
sequences). The kit used to purify plasmid is the macherey nucleospin kit,
and the primer used for sequencing is M13. We have tried to change the
primer, the lysis buffer is now prepared each day, the elution is done in
10mM tris, 1mM tris ou 0.1 mM tris with no difference...  We have checked
the amont of DNA, and it is not correlated... And it seems that the control
plasmids have no problems in the hand of the sequencing staff. Now we have
no more ideas, any suggestion will be helpful.

Thanks for all of your input in advance, and excuse my english.

Eve



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