Unltracompentent cells

Lee Vogel vogel at sb-roscoff.fr
Mon Mar 4 12:01:05 EST 1996


Have recently been mucking about with competent cell methods in order to
improve yields of a plasmid library. I started with the method of Inoue
(1990), growing XLI-blue cells to OD.600=0.6 at room temperature. After the
Mg++/Ca++ washing step I suspended the cells either in the glycerin/PEG
solution of Nishimura et al, NAR 18 (1990), or in the TB plus DMSO solution
of Inoue. Cells were then frozen in a dry-ice bath an left at -80 O.N. The
following day transformations were performed with cells preincubated with
25mM beta-Mercapto. or 25mM DTT or nothing. These transformations were
compared with supercomptent cells purchased from Stratagene which are
"guaranteed" to have a competency greater than 10-9. Cells in the
glycerin/PEG gave at least 100 fold less colonies than the Statagene and
Inoue cells. In all cases a reducing agent improved transformation, but
beta-mercapto. worked beter than DTT. Interestingly , the Inoue preparation
with beta-mercapto. gave marginally beter results than the Stratagene cells
(approx. 30% more colonies : 130 as opposed to 100 for Stratagene).
Unfortunately, I can not provide transformation efficiencies as this was a
"real" experiment involving the transformation of a ligation reaction for
which the concentration of ligated vector is unknown.





More information about the Methods mailing list