Protocol 15 - Producing Competent DH5 Bacteria for Transformation
Supplies needed:
- LB Broth
- 250 ml sterile flask
- 50 ml Falcon tube
- 10 mM NaCl
- ice cold 50 mM CaCl2
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Equipment needed:
- incubator/shaker for bacterial growth
- spectrophotometer and cuvettes
- clinical centrifuge
- wet ice bath
1. Grow an overnight culture of DH5 in 5 ml of 2X TYE Broth at 37°C without
ampicillin. (2X TYE : 20 g tryptone, 10 g yeast extract, 10 g NaCl)
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2. Innoculate 250 ml of fresh 2X TYE broth without ampicillin in a 500 ml flask
with 2.5 ml of the overnight DH5 culture.
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3. Measure the O.D. of the cells until an optimal density of 0.6-0.7 is achieved.
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4. Once O.D. is achieved, centrifuge at 5,000 rpm for 10 minutes at 4°C in a 50 ml
Falcon tube. Decant off the supernatant without disturbing the pellet of bacterial cells.
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5. Wash and then resuspend the cells using 30 ml of sterile 10 mM NaCl (first add
5 ml of 10 mM NaCl and then an additional 25 ml - be sure the cells are
resuspended.)
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6. Once again, centrifuge at 5,000 rpm for 10 minutes at 4°C in the 50 ml Falcon
tube. Decant off the supernatant without disturbing the pellet of bacterial cells.
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7. Resuspend the cells in 25 ml of ice cold 50 mM CaCl2 (1/2 original volume in
step #2 above.)
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8. Incubate the cells on a wet ice bath for 30 minutes. This step will cause the
cells to become competent to pick up foreign DNA such as plasmids and
phagemids.
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9. Once again, centrifuge at 5,000 rpm for 10 minutes at 4°C in the 50 ml Falcon
tube. Decant off the supernatant without disturbing the pellet of bacterial cells.
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10. Lastly, resuspend the cells in 5 ml of ice cold 50 mM CaCl2 (1/10 of original
volume in step #2 above.) You now have a stock of competent cells which should
be stored at 4°C prior to use.