Follow these simple tips to get superior results while transforming with chemically-competent cells.
1. Cells are best thawed on ice. DNA should be added as soon as the last trace of ice in the tube disappears. Cells can be thawed by hand, but warming above 0°C decreases efficiency.
2. For maximum efficiency, be sure to incubate on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.
3. Both temperature and time are specific to the transformation volume and vessel. Typically, 30 seconds at 42°C is recommended for cloning strains (please follow the protocol for exact times as they vary for expression strains).
4. Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in TE for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium. Incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency.
5. Selection plates can be used warm or cold, wet or dry, with no significant effects on transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
Additional transformation troubleshooting help can be found on neb.com.
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