Protocols

Easy 4 degree competent cells

Posted on Thursday, November 17, 2005

Description
A method for preparing competent E. coli for transformation. The key advantage is that these cells can be kept for a week at 4 degrees Celsius.

Procedure
1. Grow an overnight culture of E. coli XL-1 in LB with 10 mics/mL tetracycline

2. Take 4 mL of this culture and put into 76 mL of LB with 10 mics/mL tetracycline; grow for 1.5 hr (this should get it well into log phase)

3. put culture on ice, 10 mins.

4. spin 10 min. at 2,500 rpm in a clinical centrifuge or equivalent (i.e. to pellet cells)

5. decant supernatant; resuspend in 20 mL KMES buffer (see recipe below)

6. place on ice for 1-1.5 hours

7. spin again as in step 4; resuspend in 4 mL KMES buffer

This cell suspension is competent for transformation and can be kept at 4 degrees Celsius for up to 7 days.

Transformation protocol:

1. Use 300 microlitres of the above cells. Mix gently with up to 50 microlitres of ligation reaction to transform.

2. Put on ice, 45 mins.

3. Heat shock at 42 Celsius for 1.5 to 2 mins.

4. Let recover 1-2 mins. on ice.

5. Plate immediately (or if superstitious, allow to recover 30 mins. at 37 Celsius, then plate). Use whatever selection is relevant to your plasmid (usually Amp); remember that these cells are intrinsically Tetracycline resistant.

Recipes
KMES buffer:

60 mM CaCl2
20 mM KMES, pH 5.8
5 mM MgCl2
5 mM MnCl2



Supplies
KMES can be bought from Sigma.

You will also need a stock of XL-1 Blue E. coli.

Tips


Submitted by: rwintle