Can't freeze my comp cells!
David L. Haviland
dhavilan at imm2.imm.uth.tmc.edu
Wed Mar 29 09:39:36 EST 2000
> Susanne Rohrer wrote:
> > As someone else stated, the Inoue Method is fine. What we watch out for
> > is to add the DMSO very slowly, i.e. dropwise while stirring the cells,
> > on ice, on a slow shaker. Then we just snap-freeze in LN2. Thawing them
> > slowly is recommended as well.
> Just a brief note: Hanahan reported that oxidised DMSO
> affects the competence of cells, however, Inoue also
> reported oxidation of DMSO have no effect whatsoever. So it
> may behave differently for different methods?
Yea... I was confused by that too when I prepared chemical competent
cells years ago... (We now opted to go with electroporation and make
our own bugs (Sure's, Top10F', and XL-1s) and getting 10^9 per/ug is
pretty common. Getting 10^8 can be usually be had on the first attempt
at cell preparation.)
Anyway, back to the point... I noticed a difference in DMSO stocks when
it came to freezing chem-comp cells. I had lower efficiency when I used
the DMSO off the shelf that had been around and absorbed water from the
air. The transformation efficiency was greatly improved (almost back
up to "same day" prep efficiency ala Inoue) when I switched to the
individually sealed 1 ml ampoules of DMSO that I bought from Sigma. We
just broke it open used what we needed and discarded the rest.
Hope this helps,
David L. Haviland, Ph.D., Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd., Houston, TX 77030
If everything seems to be going so well, you have obviously
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