User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/01/23
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The objective of this session was to insert plasmid DNA into cells in order to transform the cells. The protocol from New England Biolabs for transformation (C2527) was followed.
Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0 degrees Celsius will decrease the transformation efficiency.
Incubation of DNA with Cells on ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.
Heat Shock: Both the temperature and the timing go the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42 degrees Celsius in optimal.
Outgrowth: Outgrowth at 37 degrees Celsius for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.