Solid-state library amplification

Michael Coady coady at ERE.UMontreal.CA
Thu Jan 6 07:08:54 EST 1994


In article <2ggraa$l9k at mserv1.dl.ac.uk> sbhattac at crc.ac.uk (Dr. S. Bhattacharya) writes:
>I am writing about a problem I encountered using  the solid-state
>library amplification method described by Michael Kriegler (Gene
>Transfer and Expression - a lab manual, Stockton Press NY), p 131.
>In this method, the bugs (containing plasmid dna), are grown in a gel matrix
>(0.3% SeaPrep agarose- LB), the theory being that colonies become nutrient
>limited in 3 dimensional space and slower growing colonies catch up with
>faster ones. The bugs are then spun out of the gel matrix and maxi-prepped as 
>usual.
>All went well until the final step. On spinning the gel matrix containing the 
>bugs, at 8000 rpm as recommended, the agarose spun out with the bugs, causing
>much despondence.  I was able to melt the agarose and recover the bugs and
>library, but what went wrong?
>I had used Boehringer agaroseMP -  not having Sea-Prep, could this be the
>cause?
	[Slight editing above]

	Well, I've had the same problem using Sea-Prep, so that won't
solve your problem.  What finally worked for me was to do short spins
(3 minutes) at 12,000 rpm in our Beckman JA-20 rotor (yes, only about
35 ml per tube).  What you are doing is a differential centrifugation,
and you get a hard pellet of cells at the bottom with the beginnings
of a slight pellet of agarose.  I pour off the supernatant and gently
rinse the tube with a bit of ddH2O and pour off the rinse.
	There is still a bit of agarose left over, but it shouldn't
be a problem.  If you are going to immediately purify plasmids from
your E. coli prep, I'd STRONGLY urge that you use the triton lysis
method that is described in Current Protocols.  I had used the regular
alkaline lysis prep and I was getting a lot of sticky gunk in my
plasmid preps.  Is it agarose or bacterial debris???  I never did
find out but by rinsing my bacterial pellet once with 0.5 M NaCl
(as one kind respondent here had suggested) and by using the Triton
Lysis method (as suggested by Carl Perez, who works with Michael
Kriegler who developed this method) the problem disappeared.
	Good luck with this method and feel free to contact me if
you have any further problems.


Mike

-- 
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.



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