Qiagen Quiaquick : reliable? pure enough for Autoseq?

[Matt Hudson]

Does anyone there notice variability in the purity and yield from the 
Quiaquick kits ('specially the gel kit). Sometimes I get nice DNA with 
80% yield and a lovely spec. with good 260:280. More often, and 
especially recently, since the tops of the little purple columns went 
flat instead of dimply, the yield is dismal and there is a funny little 
shoulder on the spec. at 220-230nm. I follow their protocol exactly each 
time, including the optional bits for purer DNA. 

My problems (read only if genuinely interested)
I am trying to sequence a genomic coding region where I reckon there is 
a single-base mutation. I've got lots of primers producing overlapping 
products from it which I am sequencing directly, using PE dye 
terminators and primers (there are PE primer sequenses at the 5' end of 
the amplimers which makes them BIG - 45bp). The trouble is not all the 
primers work that well, so I can't just remove primers and nucleotides 
and add the pcr reaction - I need to gel purify the band. Getting DNA of 
decent enough quality to do this from a gel has eluded me for some time 
- I do get sequence, but it's dodgy with short reads and highly 
inaccurate. Not what you need for spotting one different base {:-(}.

Thank you anyone out there who takes pity on me
Yours worried

Matt Hudson
Botany Dept
Leicester University
Leicester LE1 7RH
UK

tel. (UK) 0116 252 3399
meh2 at le.ac.uk