Bacterial Transformation - CaCl2 Method

Jeff Lawrence

1. Grow 5 mL culture overnight. Add 1 mL of culture to 35 mL LB in a Klett flask. Grow until Klett 140.

2. Place culture on ice for 3 min. Pour cells into prechilled SS34 tubes. Centrifuge for 10 min at 7 krpm. Discard supernatant.

3. Resuspend cells in 10 mL ice cold 30 mM CaCl2. Centrifuge as above. Discard supernatant.

4. Resuspend cells in 3 mL ice cold 30 mM CaCl2. Incubate on ice for 2 hrs to overnight.

5. Add glycerol to 10% final concentration. Quick-freeze 100-300 µL aliquots in a dry ice/ethanol bath.

6. To transform, thaw aliquot on ice. Add 10-100 ng DNA and incubate on ice for 30 min.

7. Heat shock at 42° for 45-60 sec in a water bath. Replace samples on ice.

8. Phage transformations may be plated immediately with 100 µL fresh overnight host cells. If selecting on antibiotics, add 500 µL LB and incubate at 37° for 60 min.

9. Add cells to 2.5 mL melted Top agar at 42°. Vortex and plate. Incubate overnight.

Last Update: Thursday June 19 2014
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