IGEM:Harvard/2007/week 4: Difference between revisions
(New page: '''Electroporation 7/10/07''' 1) Inoculate cells from plate or liquid culture etc. 2) Grow to an OD of 0.4 to 0.8 which corresponds to log growth. 3) Put 1ml into 1.5 ml tubes. 4) Pellet ...) |
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'''Electroporation 7/10/07''' | '''Electroporation 7/10/07''' | ||
1) Inoculate cells from plate or liquid culture etc. | 1) Inoculate cells from plate or liquid culture etc.<br> | ||
2) Grow to an OD of 0.4 to 0.8 which corresponds to log growth. | 2) Grow to an OD of 0.4 to 0.8 which corresponds to log growth.<br> | ||
3) Put 1ml into 1.5 ml tubes. | 3) Put 1ml into 1.5 ml tubes.<br> | ||
4) Pellet via centrifugation at 14k rpm (max) for 30 seconds, remove supernatant and resuspend in 1mL of prechilled water. Do this 2 times. | 4) Pellet via centrifugation at 14k rpm (max) for 30 seconds, remove supernatant and resuspend in 1mL of prechilled water. Do this 2 times.<br> | ||
5) For the third time, after centrifugation, remove supernantant and resuspend in prechilled water containing DNA at desired concentration. (50ng of DNA in 50 uL of water) | 5) For the third time, after centrifugation, remove supernantant and resuspend in prechilled water containing DNA at desired concentration. (50ng of DNA in 50 uL of water)<br> | ||
6) Transfer to prechilled cuvettes. Avoid air bubbles. | 6) Transfer to prechilled cuvettes. Avoid air bubbles.<br> | ||
7) Wipe sides of cuvette thoroughly to remove water. | 7) Wipe sides of cuvette thoroughly to remove water.<br> | ||
8) Put into electroporator: for 0.1cm gap cuvettes, 1.8kV; for 0.2cm gap, 2.5kV. | 8) Put into electroporator: for 0.1cm gap cuvettes, 1.8kV; for 0.2cm gap, 2.5kV.<br> | ||
9) Press pulse button to electroporate. Record time constants (ms) | 9) Press pulse button to electroporate. Record time constants (ms)<br> | ||
10) Immediately resuspend in liquid media (1ml). | 10) Immediately resuspend in liquid media (1ml).<br> | ||
11) Let recover in incubator for 1 hour. | 11) Let recover in incubator for 1 hour.<br> | ||
12) Plate on selective agar plates to isolate transformed colonies. Grow overnight. | 12) Plate on selective agar plates to isolate transformed colonies. Grow overnight.<br> | ||
Revision as of 13:31, 10 July 2007
Electroporation 7/10/07
1) Inoculate cells from plate or liquid culture etc.
2) Grow to an OD of 0.4 to 0.8 which corresponds to log growth.
3) Put 1ml into 1.5 ml tubes.
4) Pellet via centrifugation at 14k rpm (max) for 30 seconds, remove supernatant and resuspend in 1mL of prechilled water. Do this 2 times.
5) For the third time, after centrifugation, remove supernantant and resuspend in prechilled water containing DNA at desired concentration. (50ng of DNA in 50 uL of water)
6) Transfer to prechilled cuvettes. Avoid air bubbles.
7) Wipe sides of cuvette thoroughly to remove water.
8) Put into electroporator: for 0.1cm gap cuvettes, 1.8kV; for 0.2cm gap, 2.5kV.
9) Press pulse button to electroporate. Record time constants (ms)
10) Immediately resuspend in liquid media (1ml).
11) Let recover in incubator for 1 hour.
12) Plate on selective agar plates to isolate transformed colonies. Grow overnight.
