Editing Competent-Cells (section)

== CaCl2 Competent Cells---Do not use for cloning, maybe Ok for subcloning ==

1. Optional: From library efficient Top10 or STBL3, steak out a new plate and place at 37oC overnight.

2. Inoculate two to three colonies into 5 ml LBY and grow overnight at room temperature (23 to 25oC).

4. Add cells to 500 ml LBY.

5. Grow the cells at room temperature until they reach an OD600 of 0.6. The bacteria will double every 2 to 2.5 hours at 37 oC.

6. Prepare clean centrifuge tubes:

if not autoclavable:<br/>Wash centrifuge tubes with 10% bleach, rinse with water and Millipore water.<br/>Spray with 75% ethanol and dry in biosafty hood with fan on.

7. Transfer cells to autoclaved centrifuge tubes (450ml), and place on ice for 10 minutes.

8. Centrifuge at 3000 rpm for 5 minutes at 4oC using a pre-chilled rotor.

9. Resuspend cells gently in 30 ml of ice cold 0.1 M CaCl2, transfer to sterize/high performance 50 ml centrifuge tubes

10. Keep the cells on ice for 10 minutes.

11. Centrifuge cells at 3000 rpm for 1 min at 4oC.

12. Resuspend cells gently in 20 to 60 ml of ice cold CaGly Solution.

13. aliquote 100 ul to chilled autoclaved 100 ul tubes.

14. Freeze cells on dry ice and store at - 80 oC.

'''CaGly Solution.'''<br/>0.1 M CaCl2<br/>15% glycerol

'''Inoue Solution&nbsp;'''(10 mM PIPES, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl.):

PIPES 3&nbsp; g/l,

CaCl2.2H2O 2.2&nbsp; g/l,

KCl 18.6&nbsp; g/l,

MnCl2.4H2O 10.9 g/l&nbsp;

Mix all the components except for MnCl2, adjust pH to 6.7~7.0 with KOH.

Then, dissolve MnCl2 , sterilize the solution by filtration through a prerinsed 0.45 µm filter unit and stored at 4ºC.

<br/>'''0.1 M CaCl2.'''