Eric R. Hugo
eric at bcserv.WUSTL.EDU
Mon Jul 26 06:10:00 EST 1993
>> 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.
>Has any one tried a INSTANT competency buffer from 5-3 prime?
>They claim that you can get 1x10-6 colonies per ug of DNA starting
>from cells scraped from a plate?
>Appreciate the input.
>For those interested, 5prime-3prime is in Colorado 800-533-5703.
>I have no association with this company and all the other disclaimers.
>bsh at med.pitt.edu
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).
Eric R. Hugo, Postdoctoral Research Associate |eric at bcserv.wustl.edu
Dept. of Biochemistry and Molecular Biophysics|finger above for
Washington University School of Medicine |public key <--
Box 8231, St. Louis, MO 63110_________________| (314) 362-3342
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