Electrocompetent

Additional notes on the AdEasy system are available at www.coloncancer.org/ adeasy.htm. The most challenging step in the procedure is the generation of successful recombinants. Several factors affected recombination efficiency, including the quality of the preparation of the electrocompetent BJ5183 cells. We found that increasing the time of incubation of the cells on ice from 30 min to 1 to 2 h improved recombination efficiency. We typically isolated at least 10 colonies to ensure identification of a recombinant vector. 

Pipette 50 pL of electrocompetent HB101 cells (19), with an efficiency of transformation of at least 107 CFU/pg of DNA, into an ice-cold sterile microfuge tube and place it on ice. 

Electrocompetent JM109 Eschericha coli cells (Life Technologies, Merelbeke, Belgium). 

Chill the 20 fxg of purified DNA and a 0.2-cm gap electroporation cuvet on ice. Thaw a 350pL aliquot of electrocompetent E. coli SS320 on ice. Add the cells to the DNA and mix by pipetting several times (avoid introducing bubbles). 

The final volume of resuspended, electrocompetent yeast should be 1-1.5 mL (rrrNote 5). 

In a sterile, ice-cold Eppendorf tube, mix 40 pL concentrated, electrocompetent yeast with 10-100 ng of transforming DNA (<5 pL) (see Note 6). 

Electrocompetent yeast can be stored at -80°C for future use by adding glycerol to 15% final concentration and snapfreezing in a dry ice/ethanol bath. To use frozen cells, thaw slowly on ice, pellet the cells, and resuspended to the same volume in fresh, ice-cold 1M sorbitol. This will remove excess ions released by dead cells. The transformation efficiency using these frozen/thawed cells will drop > 10-fold from fresh yeast. 

Mixing the DNA with the electrocompetent yeast and allowing them to incubate together on ice for 30-45 min may improve transformation efficiency. 

Add 100 pL of electrocompetent cells (approximately 5 x 10 spirochetes) and 5 jL (25 pg) of plasmid DNA in a microfuge tube, mix gently, and transfer to the bottom of a 0.2-cm electroporation cuvette. Avoid introducing air bubbles in the sample that can cause arcing. Gently tap the cuvette to ensure that the sample goes to the bottom. 

Fig. 4. Virulence of strains by mouse infection. Following in vitro manipulation, strains were assessed for viralence by intraperitoneal injection of mice and subsequent microscopic detection of Giemsa-stained spirochetes in blood. Injection of equal numbers of (A) electrocompetent Borrelia hermsii DAH 2E7, (B) electroporated without a manipulation constmct or antibiotics and cloned by limiting dilution, and (C) the vtp mutant all produced comparable spirochetemias at 65 h post infection. Images of infected blood were taken at 400x.

Electroporate the purified ligation mixture into electrocompetent E. coli TGI using 2 pL Hgation mixture per electroporation (i.e., 20 electroporations in total). 

T4 DNA ligase (lU/pL) and lOX ligation buffer (Boehringer-Mannheim). Electrocompetent bacteria (e.g., DH5a). 

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). 

Dispense the electrocompetent cells in 50-100 p,l aliquots in 1.5 ml polypropylene tubes and proceed as quickly as possible to the transformation protocol (see Protocol 15). 

Transform 100 pL of supercompetent XL-1 Blue E. colt with 1-5 pL of the ligation reaction as directed by the supplier (Stratagene) It is essential to obtain high-transfection efficiency, especially when looking for rare clones I found it very reliable and convenient to purchase supercompetent cells for this purpose. If this is not possible, I recommend using electrocompetent cells and electroporation to transform bacteria (68). 

Alternatively, electrocompetent bacteria and a suitable electroporation device are required. 

Competent Escherichia colv JM109 electrocompetent bacteria (see Note 7)... 

The Gene Pulser (Bio-Rad) provides a fast, efficient way to perform the numerous bactenal transformations that must be undertaken with the differential-display technique. We prepare our own electrocompetent bacteria from frozen stocks as suggested by the manufacturer... 

The EPI300 (Epicentre) electrocompetent E. coli cells work well with this procedure. Combine 3 pi DNA with 30 pi of these cells in a 1-mm cuvette (BioRad) and electroporate the cells at 1,200 V, 25 pF, and 200 Q using a Gene Pulser Xcell electroporation system (BioRad). 

Preparation of Electrocompetent DH10B Cells Containing Desired BAC... 

Induction of the Lambda Recombination Genes and Preparation of Electrocompetent Cells... 

In a 0.5-ml tube, mix appropriate volume of DNA (200-300 ng of salt-free PCR fragment) with 50 pJ of electrocompetent induced or uninduced cells. Leave the tubes on ice for 5 min. 

Wash the pellet in 70% ethanol and dissolve the air-dried pellet into 50 pi sterile distilled water. From that, 40 pi (approximately 1 pg) can be used for restriction analysis in a 50-pl reaction, and 1 pi can be used as template for PGR analysis or for transformation into electrocompetent SW102 bacteria. 

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