Minipreps for ABI sequencing

Bernard Murray bernard at elsie.nci.nih.gov
Sat Jun 22 12:28:16 EST 1996


In article <31CB5832.1C60 at physci.ucla.edu>, ecastro at physci.ucla.edu says...
>
>AShaw5 wrote:
>> 
>> The "new" wizard kit is just a modification of the wash buffer.  There is
>> now KCl instead of NaCl and less EDTA.  We found variability with the old
>> kits, but the sequnece now looks pretty good.
>> Andrey Shaw
>
>Is this the only change?  I was under the impression that there was a 
>copyright problem with another company (Biorad?) and that they had
>been forced to change the resin itself.

No, this wash buffer change is a recent change within the Wizard kit.
After the "patenting problem" (which Promega reps deny) they shifted
from diatomaceous earth to silica gel.  This was the move from Magic
to Wizard.  They've had quite a lot of problems after this, hence all
the yellow pages in the latest methods leaflets.  Based on the various
methods sheets I've seen around here the protocol modifications after
the Wizard change are as follows;

1)	Change the ethanol concentration in the wash buffer.
They suggested a move from 60 to 50% but this resulted in a drastic
reduction in yield in our hands and their explanation that the "extra
yield" was RNA contamination turned out to be incorrect for us.  We
moved back to 60% and hung on to the better yield.

2)	Expose the resin to the crude TE preparation for a shorter time.
It seems that silica gel binds bacterial nucleases (from what I can
determine the old Magic resin did not do this).  This is particularly
nasty if you are using an endA+ strain as you will either end up with
a low yield or a preparation that requires phenol/chloroform extraction.

3)	Change the wash buffer.
There seems to be carry over of the Na+ and EDTA in this buffer and this
can inhibit downstream reactions.  Promega switch to K+ based buffer
to dodge the first problem and suggest adding extra Mg2+ to reactions
to dodge the second.  From what I have seen I think that this is all
due to the nature of the silica gel as the diatomacous earth dries
more completely when a vacuum is applied and there is no detectable
carryover of the wash buffer with the Magic resin (side by side
comparison).

Just to spell it out, I've become quite fed up with Promega's attitude
over this kit (they've been just fine in all other respects).  Their
policy has been to deny that there are any problems and blame the user.
I've wasted time/effort/money playing along with the little modifications
and at the end of the day I simply believe that the silica gel method
is inconsistent at best.  After some particularly discouraging results
during purification from HB101 (before they admitted the nuclease binding
problem) I switched to the "Merlin" diatomaceous earth method posted to
this group some time ago and have never looked back.  I don't have any
problems with Promega per se and in fact I still use their spin columns
with my reagents.
	Disgruntledly,
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)
"I don't get it.  Is it cool to make no sense, Joel?  Is it hip to be vague?"
			-Crow T. Robot, MST3k (Robot Monster)




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