Frequently Asked Questions - Acella Electrocompetent Cells

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The more DNA used, the more efficient the ligation will be. The ratio of vector to inserted DNA can be critical for obtaining high-efficiency ligations. A molar excess of insert to vector may yield higher efficiency ligations when subcloning inserts into plasmid vectors. However, an equal or greater ratio of vector to insert may be preferred when performing library construction into plasmid vectors. Before electroporation, the DNA should be diluted or precipitated as high DNA concentration in the electroporation can cause arcing.

Yes, ligated DNA does require dilution or desalting prior to transformation. For ligation reactions dilute at least 1:5 with water or TE buffer, or use a proven method for desalting before electroporation.

We recommend using 1µl DNA with 20µl cells for each transformation, larger size reactions can be used (up to 40µl in a 0.1cm gap cuvette) but the volume of DNA used should not be more than 5% of the cell volume.

These are satellite colonies. They are not transformants. Incubate your plates for less time (18 hours is good — never more than 20), use more antibiotic, or use fresher plates to get rid of them.

Because field strength is critical for bacteria, the choice of cuvette is important. In order to achieve the proper pulse, we suggest 1mm cuvettes. Yeast and fungi require 2mm cuvettes, while 4mm cuvettes are ideally suited to mammalian and human cells.

Avoiding conductive ions is a good way to avoid arcing when electroporating bacteria. This can be achieved by lowering the ratio of DNA-to-cells in the sample. Always avoid air bubbles and condensation that can accumulate on the electroporation cuvette. Dilute or precipitate ligation reactions before electroporation, carefully following washing procedures to remove salts. For ligation reactions dilute at least 1:5 with water or TE, or use a proven method for desalting before electroporation. Do not use more than 5µl total volume of DNA with 20µl cells. Using only highquality cuvettes can also reduce the chances of arcing.

Field strength is critical in electroporation. Field strength is usually expressed as kilovolts/centimeter (kV/cm), where kV is equal to the initial peak voltage and cm is equal to the size of the gap between the electrodes of the cuvette.

Proteins toxic to E.coli are better expressed in Acella™ Chemically Competent Cells containing the pLysS plasmid. The pLysS plasmid will express low levels of lysozyme that will bind to T7 polymerase, therefore inhibiting transcription. This will lower the basal expression of the protein during the pre-induction growth. If the protein is extremely toxic, then it is better expressed in a BL21 (DE3) pLysE strain or using a combination of a Acella™ Electrocompetent Cells and a T7 lac promoter.

Our Acella™ Electrocompetent Cells are designed for high-level protein expression using T7 RNA polymerase-based expression systems. Acella™ pLysS Chemically Competent Cells provides tighter control for expression of toxic proteins. Tighter control is provided by strains carrying the pLysS plasmids encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase.

The expression properties of Acella™ Electrocompetent Cells are the same as the standard BL21. It is difficult to predict because protein levels as well as solubility will vary from protein to protein. In general, long proteins are much more difficult to express than shorter proteins. Also, human proteins that contain clusters of codons rarely used in E. coli may have a tendency to give lower yields and/or truncated products.

Yes. With the exception of the added ΔendA ΔrecA genotype, the Acella™ Electrocompetent Cells are identical to standard BL21. BL21 cells naturally lack ompT and Lon proteases improving stability of synthesized proteins. BL21 (DE3) expresses T7 polymerase after induction with IPTG, therefore regulating the transcription of genes under the T7 promoter (for example, those in pET plasmids).