Toronto/Lab Protocols/Transformation

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Revision as of 03:30, 27 October 2007

Transformations

4 hours

Transformation is a genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). ([http://en.wikipedia.org/wiki/Transformation_%28genetics%29 Wikipedia])

Competent cells are bacteria, which can accept extra-chromosomal DNA or plasmids ([http://en.wikipedia.org/wiki/Competent_cells Wikipedia]).

For each plasmid:

1. Take competent cells from –80 °C freezer.
2. Leave on ice for 10 minutes.
3. Add 2-5 μL DNA to competent cell. Remember to label.
4. Mix with pipette.
5. Sit on ice for 30 minutes.
6. Get a beaker of exactly 42 °C water from hot water tap.
7. Submerge tubes in hot water for exactly 90 seconds.
8. Sit on ice for 5 minutes.
9. Add 1 mL of LB.
10. Incubate for 1 hour.
11. Spread 1 mL on a Petri plate labeled with the appropriate antibiotics. (Dip a glass rod in ethanol and flame it to sterilize, then spread the cells on the plate.)
12. Leave the plate with lid half open in the incubator to dry. Rotate the plate 180° every 10 minutes to ensure even spread of bacteria.
13. Once dry, turn the plates upside down and incubate overnight.

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