Does anything grow from these on plates with no selection?
A conservative thing to try might be to remove a large chunk of the
frozen stock and grow it in LB medium without selection for an hour
or so... maybe this would give any viable cells a bit of time to
recover if they weren't frozen properly. Then spin down the culture
and steak the whole cell mass onto a selective plate-- you would only
need one viable cell with the plasmid.
Can I assume that you don't have a stock of the plasmid prep to
retransform? I am usually far more careful about keeping a plasmid
preps for all constructs than glycerol stocks, since if prepped
right, plasmid preps would be perfectly fine at room temp for quite
some time.
Mike
-----Original Message-----
From: methods-boun...@oat.bio.indiana.edu [mailto:methods-
boun...@oat.bio.indiana.edu] On Behalf Of Sunil Dalvi
Sent: Wednesday, March 24, 2010 1:51 AM
To: meth...@magpie.bio.indiana.edu
Subject: plasmid from non viable glycerol stock
Hello,
I am working with Agrobacterium transformation of sugarcane.I
prepared the
glycerol stock of plasmid transformed in E.Coli DH5 alpha and EHA
105. After
some months those are non reviving. What may be the problem? Is
there any
method to recover plasmid from glycerol stocks.
Please help me
Is there anybody who can help me
--
S.G.Dalvi,
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)
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