1) Use a 30-mer sequencing primer (i.e. longer than the inverted repeat
primer), to make it compete more successfully for the template with the
other end of the product.
2) Use higher (3x-10x higher) concentration of the sequencing primer and the
lowest possible concentration of the PCR product, again with the aim from
"1".
3) In a ThermoSequenase (or similar) thermal sequencing reaction, increase
the annealing temperature from the recommended 55 deg to 60-65 deg.
Good luck,
wagner
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Thomas J. Stelick
Sent: 11 aošt, 1999 13:04
To: Recipients of ABRF List
Subject: dna seq
We are trying to sequence a cloned PCR product for a customer but we are
having a problems. The pcr product has a 24 base inverted repeat at the
ends which is causing to fold on itself and the sequence can not get
through this structure. We have tried DMSO but no difference is seen.
Does anyone have any suggestions on how to get through it.
Tom Stelick
TJS11@cornell.edu
BioResource Center, Cornell University
157 biotech bldg.
Ithaca, NY 14853
(607)254-4857
http://brcweb.bio.cornell.edu