Freezing of the cells and efficiency of transformation.
malexeyev at biost1.thi.tmc.edu
Tue Dec 12 10:46:35 EST 1995
In article <4ak1g7$6e5 at snowball.hscsyr.edu>, Tom Duncan
<duncant at cross.bmb.hscsyr.edu> wrote:
> We also use Inoue's 1990 method (with growth at up to 22oC) with
> results of 5x10^7 to 2x10^8 cfu/ug for most cloning strains. The best thing
> seems to be that efficiency actually increases after the initial freezing, and
> aliquots of frozen cells (-70oC) maintain a stable efficiency up to 3 months.
That is an interesting observation that efficiency is really increasing
after initial freezing. I observed a strange effect related to this: I
have three essentially isogenic strains one is being TG1 (or at least I
received it as TG1), another is same, but bearing the gene for EcoRV
methylase on the plasmid and third having the same methylase gene on a
transposon in the chromosome. I prepared competent cells of all three
strains using protocol described in Chung, Miller Meth. Enzymol. v 218
p.621. In a few month after the freesing efficiency of the TG1
transformation dropped while that of other two strains increased appr.
3-10 fold. Does anyone have any explanation to the effect?
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