Paul N Hengen
pnh at fcs260c2.ncifcrf.gov
Tue Jul 27 11:16:24 EST 1993
In article <230e28INN3lc at medicine.wustl.edu> eric at bcserv.WUSTL.EDU (Eric R. Hugo) writes:
>>> I am preparing competant cells for the first time and have chosen the
>>> "one-step" approach referenced above because it appears to be the most
>>> convenient. I will be using E. coli strain HB101 and vector pBR322
>>> (containing a 6kB insert). I would appreciate any hints from those who use
>>> this method.
> D. Hanahan (J. Mol. Bio (1983) 166:557-80) describes a method
>for colony transformation that works well in my hands. Efficiency
>is not all that high but the method is very useful for moving a con-
>struct between strains. My modificaation to Hanahan's protocol
>is simply to resuspend 4-5 colonies in 200 ul of frozen storage
>buffer add 7 ul high quality DMSO, incubate on ice 10 min, add
>another 7 ul DMSO, add .2 - 1 ug miniprep DNA, incubate on ice 10
>min. Spread 50 -100 ul on selective media. Typically I get somewhere
>between 30-200 transformants. Its very quick and dirty but saves
>you the trouble of maintaining bunches of frozen competent strains.
>For ligations I always use high efficiency (10^7-10^8 transformants/ug)
>frozen competent E.coli (usually DH5 or XL1-blue). The rx for frozen
>storage buffer is:
> 10 mM potassium acetate
> 100 mM potassium chloride
> 45 mM manganese chloride (MnCl2 4H2O)
> 10 mM calcium chloride (CaCl2 2H2O)
> 3 mM hexylamine cobalt trichloride [Co(NH3)6]Cl3 (Sigma)
> 10 % redistilled glycerol
>Make the solution using 1 M potassium acetate that has been adj. to pH 7.0.
>Adjust the pH of the solution to pH 6.4, filter sterilize and store at -20 C
>According to the paper some strains may require RbCl. I've used it with the
>following strains of E. coli: W3110, MG1655, BL21(DE3).
By the time you've read this, you could have inoculated a culture, grown it
to log phase, collect a few ml of cells, resuspended in cold 75 mM CaCl2 and
transformed right away. With HB101 and pBR322, you could get away with almost
Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic
resistance in bacteria: genetic transformation of Escherichia coli by
R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.
Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium
chloride improves the competence of Escherichia coli cells. Gene
Hanahan, D. 1985. Techniques for transformation of E. coli, p.109-135.
In: D. M. Glover (ed.), DNA cloning: A practical approach, Volume I, IRL
Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
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