Rubidiumchlorid competent E.coli cells

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!!!Important!!! For making Rubidiumchlorid competent E.coli cells you already need one sample of competent cells frozen at -70°C.

These cell are used for the E coli Chemical Transformation protocol.

Generation -70°C Rubidiumchlorid competent E.coli cells stocks for chemical transformation

This is a protocol for 200ml medium which yields enough cells for 100 to 200 transformation. If cells for fewer transformations are needed feel free to scale down.

  1. Prepare 2 ml preculture with -70°C competent cells in LB and grow cells by shaking at 37°C for 14 hrs.
  2. Dilute 2 ml of fresh O/N culture in 200 ml LB + add 2 ml of 1M MgCl2 and 1M MgSO4 to get to a final concentration of 10 mM and grow cells until they reach the OD of 0,6-0,7 at 600 nm.
    CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!
  3. Place the cells on ice for 20 min and cool down the glaspipettes and Eppis at -20°C.
  4. Spin down the cells for 8 min at 4500g at 4°C. Remove supernatant.
  5. Resuspend the cells in 66 ml (1/3 of the volume) precooled RFI-solution at 0°C on ice and leave them for 30 min in the cold room.
  6. Spin down the cells for 8 min at 3000 rpm at 4°C.Remove supernatant.
  7. Resuspend cells in 16 ml of precooled RFII-solution and leave them for 30 min on 0°C.
  8. Freeze cells in 50 or 100 µl Aliquots with liquid Nitrogen.
  9. Store aliquots at -80°C immediately.
  10. Cryopreserved cells can be stored at -80°C for at least 12 months.
    To use, thaw the cells on ice.

RFI Solution (RFI)

Storage of RFI and RFII should be always at 4°C.

Ingredient Amount per 500 ml Final concentration
Rubidiumchlorid 6.0 g 100 mM
MnCl2 x 4H2O 4.95 g 50 mM
Potassium Acetate (1 Mol pH 7,5 ) 15.0 ml 30 mM
CaCl2 x 2H2O 0.75 g (2%) 10 mM
Glycerol 75.0 g 15 %

To adjust the pH of RFI use 0,2 M acetic acid and set the pH to 5,8. Afterwards the solution has to be filtered sterilly.


Procedure of making 1 M pH 7,5 Potassium Acetate:

  1. Weigh in 9,81 g and dilute it in 100 ml H2O.
  2. To adjust the pH use glacial acetic acid. Be careful, one drop is enough.


RFII Solution (RFII)

Ingredient Amount per 500 ml Final concentration
MOPs 0,5 M stock 10 ml 10 mM
Rubidiumchlorid 0.6 g 10 mM
CaCl2 x 2H2O 5.50 g (2%) 75 mM
Glycerol 75.0 g 15 %

To adjust the pH of RFII use NaOH and set the pH to 5,8. Afterwards the solution has to be filtered sterilly.


Procedure of making 0,5 M pH 6,8 MOPs:

  1. Weigh in 10,46 g and dilute it in 100 ml H2O.
  2. To adjust the pH use NaOH.