Qiagen Quiaquick : reliable? pure enough for Autoseq?

futers at biovax.leeds.ac.uk futers at biovax.leeds.ac.uk
Fri Aug 23 11:09:47 EST 1996


In article <4vkgbf$289 at falcon.le.ac.uk>, Matt Hudson <meh2 at le.ac.uk> writes:
>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
>
>

Hi Matt
I have not had problems with Qaiex II or the Qaiquick PCR kits.  But I get
very poor yields using the Qaiquick gel extraction kit.  I do not know 
why this kit does not work in my hands when the other two do as it 
is a hybrid of the other two.  I have therefore been using the Qaiex II
kit to clean PCR product for dye terminator sequencing on an ABI even 
though this kit is not recommended due to the risk of carrying over the  
glass resin.  But if you are careful taking off the supernatant carefully
it seems to work OK.
       Sorry I can not be more helpful.
                Simon Futers.




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