Sequencing problem. Is it the GeneClean?
nash at nrcbsa.bio.nrc.ca
Wed Feb 9 09:47:07 EST 1994
In article <94039.133242NORVALC at qucdn.queensu.ca>,
<NORVALC at QUCDN.QueensU.CA> wrote:
< pcr sequencing woes deleted for bandwith conservation >
>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?
Just a couple of "me too" observations.
In the past two years, I have had some problems with GeneClean II's
NaI solution. I had one batch go "off" which Bio 101 replaced, and
troubles with another batch (which was a year old, so I couldn't
complain). I have since stopped using NaI and changed to sodium
perchlorate (I believe it's 6 M... I haven't made it up, I steal it
from the lab up the hallway (thanks, Warren), or use it from a bottle
from a Schleicher and Schull kit lying around). Now the procedure is
Bio101 say that the pH of NaI is critical, or binding is reduced. All
I know is that in the last two years, my NaI is prone to turning
yellow (even though I aliquot it when I open it), and once it's
yellow, my DNA becomes crap.
Also, when I remove the perchlorate or the NEW (or whatever) from the
glass matrix:bound DNA, I aspirate it off with a drawn-out capillary
attached to a vacuum line, so carry-over is reduced.
John Nash (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences, National Research Council of Canada,
Yet another Aussie-in-exile ;-)
*** Disclaimer: All opinions are mine, not NRC's! ***
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