Sequencing problem. Is it the GeneCl

Bonham, Peta pbonham at pbi.nrc.ca
Wed Feb 9 11:57:44 EST 1994


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We found that if you dry the glassmilk pellet after the 3 NEW washes (about 5 
min in a speed vac) it increased the yield of DNA.

Peta Bonham-Smith
Biomira Inc.
Saskatoon
pbonham at pbi.nrc.ca

>   Now I wonder if my problem is in the DNA cleanup stage.  Normally I
harvest
>  a PCR band from a Nuseive agarose gel and use GeneClean II to clean up the
  
>  DNA.
> I dissolve the agarose with the NaI, add glassmilk, use NEW wash to clean
the
> DNA three times, and resuspend the DNA in a small volume of TA.
> 
>   My question is straightforward:  Have anybody else noticed that there are
> critical steps in the GeneClean protocol?  If so, what should I be focusing
on?

It may depend on what size your PCR product is.  GeneClean has various
yields depending on the size of your product.  Generally, the bigger the
better.  I believe yields are good with anything over 1 kb, dropping off
significantly under 500 bp.  I have used GeneClean for 40 bp pieces without
paying attention to the yield.  It works with fragments that small but I
bet the yield is well below 50%.  If your PCR products are in the 200-500
bp range you may be getting variable yields from GeneClean which may lead
to variable sequencing results.

Hope this helped!

-- 
Steve "Some day I will get the hell out of Wisconsin" Rodems

"You know, the other day I ... oh, wait, that wasn't me"





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