[Drosophila] Sequencing woes

sandra schulze via dros%40net.bio.net (by heterochromatic from gmail.com)
Sun May 30 11:59:07 EST 2010


Hello Flyland,

I am having a rather upsetting run of bad sequencing karma, and was
wondering if I could put out a request for recommended sequencing core
facilities. I won't mention which facility I currently use, because it
could still be my fault, but i never had as much bad luck as I am
having now.

I use 5 prime's  Perfectprep spin minikit (cat # 2300110)
I elute in water
I cut, run and spec the samples before sending off
I DO use a nanodrop UV spec which I have been told overestimates
concentration relative to cybergreen methodologies, and try to adjust
by a factor accordingly, since the current facility uses cybergreen.

My primer annealing temps are usually pretty high - 55+, and this is
currently my only explanation for the current run of failures. The
current facility I use runs all reactions with an annealing temp of 50
degrees, and an extension temp of 60 degrees.

I would be most grateful for a recommendation of a sequencing core
from which robust results are typical, and  to which I can fedex my
samples. I'll have to pay out-of-house prices, but i really need to do
some comparison shopping now.

Many thanks,

Sandra

Sandra Schulze
Assistant professor
Dept. Biology
Western Washington University
Bellingham, WA



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