Making Competent E. coli

Buffer

TfbI, pH5.8

            500ml             1.47g KOAC

                                    4.95g MnCl₂

                                    3.73g KCl

                                    0.74g CaCl₂

                                    60ml Glycerin

To make: mix, pH down to 5.8 with diluted (about 0.2M) aectic acid, sterile filter, keep at 4 ℃.

TfbII

500ml              10.47g Mops, dissolve in 300ml distilled water, adjust pH to pH7.0 with 10M NaOH

                                    then add

                                    5.51g CaCl₂

                                    0.37g KCl

                                    60ml Glycerin

Sterile filter, keep at 4 ℃.

Protocol       

-Grow single fresh colony of desired strain (see below for possibilities) for  2 hours in 5ml of LB. Shake vigorously at  37 ℃. (300rpm in orbital shaker).

-Transfer to 100ml fresh LB broth. Shake for a further 2-3 hours till OD550 = 0.5. (DH1, JM101 takes ~2 hours; HB101, DH5aF takes ~2.5 hours)

-Spin cells in two 50ml Falcon or Corning disposable centrifuge tubes at 2.5

 Krpm for 5min at 4℃. (Just enough to pellet the cells).

-Drain cells and resuspend in 20-40mls per 100mls starting culture of TfbI.

 You can be quite vigorous resuspending the cells at this point.

-Leave on ice. This step is the most critical for maximum efficiency. For  DH1 the optimal time is 5-10min. For HB101 and DH5aF it is 90-120min.

 Most other bugs should be assayed for optimal time. JM101 works pretty well if incubated for 20-30min on ice but I have not optimized it. For plasmids I see no reason to use any other bugs than HB101 which works extremely well with this procedure.

-Spin the cells as gently as possible, at 2Krpm for 5min at 4℃.

-Resuspend in 4mls per 100mls starting culture of TfbII. Be gentle and  patient. Then aliquot (0.1mls is appropriate) om sterile 5ml snap top tubes.

 Place in convenient holder and throw into liquid nitrogen. Store in -70℃ freezer.