Electroporation competent cells

For library construction it is absolutely necessary to use electrocompetent cells since electroporation provides a method of transforming E. coli to efficiencies greater than available with the best chemical methods. Using the protocol described below for preparing electrocompetent E. cdi TGl strain we routinely obtain 10 to 10 transformants/pg of DNA. Alternatively E. cdi TGI strain electroporation competent cells with a transformation efficiency greater than 1 X 10 transformants/pg can by purchased from Stratagene (200123). 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Because JC8679 competent cells for electroporation are not commercially available, we had to produce our own. We routinely prepare competent cells following the protocol reported previously (14). Although JC8679 competent cells for electroporation of 10 -10 cfii/pg pUC19 are routinely prepared, the apparent transformation efficiency for the ORF trap is much lower (typically 50-100 colonies per 30 pL of transformation culture). 

Transformation is done by electroporation of 71-18 or DH5oi cells. According to our experience, the use of chemically competent cells is not recommended, because it gives much lower yields of transformants. 

Add 20 ptL of the ligation reaction to each of the 12 aliquots of competent cells and incubate for at least 30 min on ice, then transfer into pre-chilled electroporation cuvettes. 

Make a transformation test before creating the library, by electroporating 100 pL of competent cells with 1 pL of the ligation mix, and compare the transformation efficiency with that of a standard plasmid like pUC18. For reaching high library sizes, each transformation should yield between 106 and 107 transformants. Repeat electroporations to reach the desired diversity. 

For electroporation ELECTROMAX DH10B competent cells with pUC119 control plasmid (Gibco-BRL). We use Gene Pulser (Bio-Rad, Richmond, CA) equipped with Pulse Controller and E. coli Pulser cuvet with 0.1-cm gap (Bio-Rad) for electroporation of E. coli. 

Generally, 10-20 pg of transposon plasmid DNA is alcohol-precipitated, washed with 70% ethanol to remove salts that may cause arcing during electroporation, and resuspended in a small volume (e.g., 5 pL) of sterile HjO. The final volume of DNA must be small relative to the volume of competent cells, again to avoid arcing. 

XI Preparation of electroporation competent E. coli TGI strain cells... 

The next step in cloning is the transformation of competent Escherichia coli host cells. These can be prepared in-house through electroporation or chemical treatments (e.g., calcium chloride or rubidium chloride) or ordered from a vendor separately or as part of a cloning kit. The least expensive approach is to prepare the competent cells in-house, but this involves a trade-off in time. The expense of vendor-purchased cells can be reduced by using half the recommended volume per transformation, which works fine unless there are too few transformants recovered. (For example, we have had this experience using the TOPO-based veetor and long PCR product inserts.) The manufacturer s transformation instructions can be followed directly. For the transformation of chemically competent One Shot E. coli, this entails the following steps, for a half-volume transformation ... 

If transformation with Z-Competent cells is unsuccessful, traditional heat-shock transformation or electroporation with a different brand of competent cells may yield higher efficiencies. 

Three microliters of the donor DNA solution and 1 pL of the trap vector solution are blended with a 20 pL of competent JC8679 cells for electroporation see Note 10), and the mixture is transferred into an electroporation cuvette with a 0.1 cm electrode gap. After an electric pulse of 1.67 kV is applied, 1 mL of SOC medium is added to the suspension, and incubated at 37°C for 3 h with vigorous shaking. This process is applied to each gene one by one. 

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