Competent cell preparation

Lin-Cereghino, J., Wong, W.W., Xiong, S. et al. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques, 38 (1), 44, 46, 48. 

Recipient B. subtilis strain 168 cells in sterile saline— These cells are conveniently provided as thawed 0.6-ml aliquots of frozen competent cells prepared as described in Experiment 20. You will require approximately 250 ml of these cells. 

Detomine the efficiency of transformation of each competent cell preparation by transforming the cells ivith 1 ng of uncut (supercoiled) vector DMA (pUC19 for example). This is carried out in parallel with transformation of competent cells with ligated DMA (see Protocol 13) for the library construction (proceed to Protocol 15). 

Harvest the cells by centrifugation and wash once with 25 mL of sterile water Resuspend the pellet and wash once in 5 mL LiOAc/TE. Resuspend the final-cell pellet in 1.0 mL of LiAc/TE solution and store on ice This will yield a final competent-cell preparation of approx 1.5 mL. 

The density of yeast cells in culture can be estimated spectrophotometrically by measuring the optical density at 600 nm The culture should be diluted to an ODgoo < 1.0 U and then, each 0.1 OD qq U is roughly equivalent to 3 x 10 cells/mL. For example, an initial OD qo of 0.7 units for the 50-mL culture at the start of this procedure would be equivalent to 2.1 x 10 cells/mL or a total of 1x10 cells Thus, the final competent cell preparation is resuspended to a final volume of 0 5 mL in IX LiAc/TE. 

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). 

The spheroplasts are resuspended in 2 ml of STC by very gentle stirring with a 1-ml sterile pipette tip. The competent cells should be used immediately at best or may be stored for up to 5 hr. The above preparation should be sufficient for 20 transformations. 

Table V-6 Preparation of E. coli and Yeast Competent Cells...

M CaCl2. Sterilize the solution by filtration through a Nalgene filter (0.45-p, pore size). It is convenient to store alM stock solution of CaCl2 (made in Milli-Q water) in 10-mL aliquots at -20°C. When preparing competent cells, thaw an aliquot and dilute it to 100 mL with pure water. 

Competent bacterial cells prepared as described in Subheading 3.6.6. 

Prepare a plate and a vial of One Shot competent cells (stored at -80 °C) for each sample. 

Transform 5 p.L of DNA prepared in step 2 into max efficiency DH5a E. coli competent cells, spread entire transformation reaction onto LB-Amp agar plates and incubate at 37°C overnight. 

A. Preparation of Cells. In order to facilitate the uptake of plasmids into bacteria, cultures are treated to make them more permeable, or competent . Competent cells may be purchased commercially and may be preferable for critical cloning steps as they are prepared using methods that generally give better uptake than the one given here. The method provided here (modified from Sambrook et al. 1989) is cheap and simple, does not use obnoxious chemicals such as dimethylsulfoxide (DMSO), and works with a wide variety of bacterial strains. We use it fbr routine maintenance of our plasmid stocks and for general subcloning. 

Using pure halohydrin dehalogenase (HheC), competing activities observed in whole-cell preparations were eUminated and halohydrins could be resolved via enantioselective ring-closure with excellent enantioselectivities yielding enantiomeric epoxides and nonreacted halohydrins (Scheme 2.237) [1846]. 

Transform 0.5-1 nL (0.01-0.5 ng) of sequence verified plasmid DNA (or the empty cloning vector, as required by the experiment) into freshly prepared E. colt competent cells (fccNote 5) and plate on the appropriate selection media. Incubate at 37 °C overnight (12-15 h) then store the plate at 4 °C for up to 1 week until needed for the next step. 

For preparation of competent E. coli cells, we routinely use the procedure described previously (15). In this procedure, E. coli cells are grown at 18°C prior to harvesting and preparation of the 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). 

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