Sauer:RbCl competent cells

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Rubidium Chloride Competent Cell Protocol

Solutions to be prepared in advance:

TFB1 - typically prepare 1L
30mM potassium acetate
10mM CaCl2
50mM MnCl2
100mM RbCl
15% glycerol
Adjust pH to 5.8 with 1M acetic acid. Filter-sterilize (do not autoclave) and store at room temperature or 4°C. The solution must be chilled on ice prior to use.

TFB2 - typically prepare 200mL
100mM MOPS or PIPES (pH 6.5)
75mM CaCl2
10mM RbCl
15% glycerol
Adjust pH to 6.5 with 1M KOH. Filter-sterilize (do not autoclave) and store at room temperature or 4°C. The solution must be chilled on ice prior to use.

Due to the volumes used, it is typically convenient to prepare at least a 5x larger volume of TFB1 than TFB2.

Sterile 50mL conical tubes may be used for the centrifugation steps. If reusable centrifuge tubes are used, they should be cleaned thoroughly, rinsed with a small amount concentrated HCl to hydrolyze any contaminating DNA, rinsed extensively with distilled water, and autoclaved prior to use.

Protocol

  1. Inoculate a 2.5 – 5mL culture of LB. Incubate overnight at 37°C with shaking.
  2. Subculture the overnight 1:100 into 250mL of LB supplemented with 20mM MgSO4. Grow the cells at 37°C, with shaking, to an OD600 of 0.4 – 0.6.
  3. Pellet the cells by centrifugation at 4500rcf for 10 minutes at 4°C.
  4. Gently resuspend the cell pellet in 0.4 times the original culture volume of ice-cold TFB1; i.e., for a 250mL subculture resuspend in 100mL TFB1. The cells must be kept on ice from this point on.
  5. Incubate resuspended cells on ice for 5min at 4°C.
  6. Pellet the cells by centrifugation at 4500rcf for 5min at 4°C.
  7. Gently resuspend the cells in 1/25th the original volume of ice-cold TFB2; i.e., for a 250mL subculture, use 10mL TFB2.
  8. Incubate cells on ice for 15-60 minutes, then aliquot 100µL per tube and flash freeze in liquid nitrogen. Store at -80°C.
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