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Electrotransformation of E. coli With M13 DNA

Lisa Starr and William D. Huse
Ixsys, Inc.
3550 General Atomics Court
San Diego, California 92121


Introduction
M13 vectors have been used extensively for subcloning mainly because the abundant, easily isolated single-stranded form of the mp series M13 phage DNA is a superior starting material for sequencing and in vitro mutagenesis. In addition, the blue/white selection process afforded by the LacZ fusion cloning scheme allows easy detection of recombinant phage over background. During the process of evaluating and optimizing different techniques of transforming E. coli with M13 DNA, including conventional chemical methods and electroporation, we found significant inhibition of E. coli XL1-Blue/M13mp19 transformation efficiency by the addition of routine amounts of X-Gal* and IPTG** to electroporated cells. This inhibition is not observed with plasmid or conventional transformations. The problem was circumvented by adding the X-Gal and IPTG to the bottom agar, or by allowing the cells to recover for 20 minutes before the addition of X-Gal and IPTG. Electroporation proved to be the most efficient technique for transforming these cells with M13 DNA.


Materials and Methods
Electroporation Competent Cells
Fresh streaks of XL1-Blue1 from a frozen glycerol stock were grown overnight at 37 C on L-broth (1% tryptone, 0.5% yeast extract, 170 mM NaCl2; pH 7.5) agar plates containing 10 g/ml tetracycline. Five or six single colonies were dispersed in 1 ml SOB medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM each MgCl2 and MgSO4 2; pH 7
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Recommend to use at up to 5000X dilutions for cell culture (e.g. Add 1 ul to 1 ml of culture medium.)...
Prepared in distilled water....
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