problem with sequencing in big dye v3

Eve evetoulza at NOSPAMhotmail.com
Wed Oct 16 00:50:17 EST 2002


>
> Try to repurify your plasmids or change a kit for plasmid purification. If
it does not work, try to optimize conditions for PCR (change a temperature
for a denaturation step and an annealing). Be sure, that big dye kit is the
best option for your DNA.
>

We have already tried promega and qiagen minipreps kits, with same bad
results. In fact, not all the preps give bad results, between 20% and 80% of
them are nice, depends on what... whe don't know. Next week I will try to do
some PCR reactions in the lab with more initial denaturation time, more
cycles, and increased annealing temperature . Next minipreps will be eluted
in nuclease free water, and the PCR products will be purified with EDTA. I
really hope i could obtain significant upgrade in the quality of the
sequences.
The plan to buy for the next time a big dye v3.1 (we use the 3.0).

thank you for your advices.

eve





More information about the Methods mailing list