problem with sequencing in big dye v3

Wolfgang Schechinger hubahopp at gmx.de
Fri Oct 11 08:54:05 EST 2002


Eve, 
suppose there is a problem either with pcr, vector or bugs.

can you do restriction digests with your preps? How do your preps look on
agarose gels?
are the control plasmids prepared the same way as your est vectors are?
are you able to clone and sequence unrelated pcr products?

Wo

At 08:52 11.10.2002 +0200, Eve wrote:
>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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