MAXIMUM EFFICIENCY COMPETENT CELLS

  • Rubidium Chloride (Fool Proof) Method

    SOLUTIONS

    TfbI 
    10 mM   Potassium Acetate  (MW 98.15)          1.47 g
100 mM  Rubidium Chloride  (MW 120.9)          6.05 g
10 mM   Calcium Chloride  (MW 147.2)          0.735 g
50 mM   Manganese Chloride  (MW 197.9)          5.0 g
15%     Glycerol                                 75 ml
        Water                              to   500 ml
  • Set pH to 5.8 with dilute acetic acid
  • Filter Sterilise
  • Store at 4oC

    TfbII 

    10 mM	 MOPS  (MW 209.5)                        0.21 g
75 mM	 Calcium Chloride  (MW 147.2)             1.1 g
10 mM	 Rubidium Chloride  (MW 120.9)           0.12 g
15%      Glycerol                                  15 ml
         Water                               to   100 ml
  • Set pH to 6.5 with 0.1 N KOH
  • Filter Sterilise
  • Store at 4oC

    PROCEDURE

    1. Grow a 10 ml overnight culture of XL1-Blue in T-Broth.
    2. Add 5 ml of the overnight culture to 50 ml of L-Broth in a 1 liter flask and shake at 37oC for 1 hour. At the same time pre-warm a 2 liter flask containing 500 ml of L-Broth to 37oC.
    3. After 1 hour transfer 25 ml from the 50 ml culture to the pre-warmed 500 ml L-Broth and incubate at 37oC and 300 to 320 rpm. Take an aliquot to read the OD550. Use L-Broth to blank.
    4. Read the OD550 at the end of 1 hour to determine the speed of cell growth. The cells should double every 20 min. Stop the culture when the OD550 is between 0.48 and 0.6.
    5. Transfer the culture into 4 250 ml Sepcor bottles and chill on ice for 15 min. [EVERY STEP FROM THIS POINT SHOULD BE KEPT CHILLED].
    6. Cool the GSA rotor down to 4oC in the RC-5 centrifuge.
    7. Spin the culture in the GSA rotor at 5000 rpm and 4oC for 5 min.
    8. Pour off the supernatant, aspirate off any droplets of media and wipe excess broth off the sides of the Sepcor bottles with sterile gause pad.
    9. Resuspend each pellet, very gently, in 10 ml of chilled TfbI. Keep on ice while resuspending. When well suspended add an additional 40 ml of chilled TfbI to each bottle.
    10. Incubate on ice for a further 15 min.
    11. Spin at 2000 rpm in the GSA rotor at 4oC for 5 min.
    12. Resuspend extremely gently each pellet (smear of cells) in 4 ml of well chilled TfbII and keep on ice (total of 16 ml).
    13. Place 72 microfuge tubes in a dry ice/ethanol bath. Quick freeze 220 ul aliquots of the cells and store at -70oC.

    Sreelekha Devarayalu
    December 1996