( frozen stock; 2) cells must...)Electrotransformation of E. coli With M13 DNA

frozen stock; 2) cells must be kept scrupulously near 0 C during preparation as well as during electroporation; 3) growing cells must be added to the electroporated cells immediately after electroporation; 4) DNA should be added in a small volume of water to keep ionic contaminants at a minimum (ions interfere with the electroporation process and also increase the chance of high voltage arcing in the sample); and 5) growing cells must be in good condition, preferably before stationary phase. Each batch of electroporation competent cells seems to vary somewhat in efficiency relative to field strength; we routinely titer each batch accordingly (Figure 1).

We found a decrease in the number of M13 vector transformant plaques obtained from electroporated XL1-Blue cells treated with X-Gal and IPTG which does not appear to be due solely to the presence of the X-Gal solvent, DMF. The suppressive effect varies with the amount of X-Gal and IPTG added to the electroporated cells, but is minimized when the reagents are added in a small volume or to the bottom agar only. The highest electro-transformation efficiencies of XL1-Blue with M13-derived vectors, however, are always obtained when X-Gal and IPTG are avoided entirely. We found no significant X-Gal/IPTG sensitivity of XL1-Blue electro-transformations with plasmids (data not shown). We have not fully investigated the effect with other strains of E. coli, but preliminary work with INV1αF' shows only limited sensitivity to X-Gal and IPTG. We have not been as successful obtaining high electro-transformation efficiencies (>10⁸/g) with this strain.

There is good motivation for using X-Gal and IPTG when subcloning into the M13 mp series vectors because
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