E. Coli Competent Cells

Rubidium Chloride method for

Transformation Competent E. coli

 

Procedure

1. Inoculate 200ul to 1000 ul from overnight culture into 100-500 ml Psi broth  (scale up or down as needed). Incubate at 37 C with aeration to A600=0.6-0.7

2. Ice 15 min.  From this step onward the cells must remain COLD. (4C or on ice)

3. Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)

4. Discard supernatant and add 0.4 volume (ie of original volume, here it is 40-400 ml) TfbI, resupend and ice 15 min.

5. Pellet cells as in #3.

6. Discard supernatant and resupend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 100ul  to 200ul aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.

I typically transform 50-100 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.

Medium and Buffers: