Making RuCl2 Competent Cells (DH_)

  • Making Competent Cells
  • Solutions
  • Transformation

    Protocol: Making Competent Cell Aliquots

    1. Grow a single colony in 55mL LB Broth @ 37C until Abs(550) = 0.22-0.50.
    2. Immediately put on ice for 20 minutes.
    3. Spin 50 mL culture at 7k rpm @ 4C for 7 min.
    4. Deacnt the sup and resuspend cells in 20ml of ice-cold TBI. (note: resuspend in 1ml of TBI and then add the rest)
    5. Mix well by hand and put on ice for 5min.
    6. Spin at 7k rpm for @4C for 7 min.
    7. Resuspend cells in 2ml of ice-cold TB2.Mix well and keep cool in ice-water for 15 min.
    8. Aliquot 0.1ml per eppe tube in dry ice. Store @ -80C.

    Solutions
    TB1 (100mL)
    Final Concentration Amount Needed
    100mM RuCl 10ml 1M RuCl (or 1.2g)
    50mM MnCl2 5ml 1M MnCl2
    30mM Pot. Acetate 0.294g KOAc
    10mM CaCl2 1 ml 1M CaCl2
    15% Glycerol 30ml 50% glycerol
    Sterile Water ??mL Water (to 100ml)
    • Adjust pH to 5.8 with 0.2M acetic Acid.
    • Filter w/ 0.45um and Store at -20C. (Pre-Rinse filter with sterile water)
    TB2 (100mL)
    Final Concentration Amount Needed
    75mM CaCl2 7.5ml 1M CaCl2
    10mM RbCl2 1ml 1M RbCl2
    10mM MOPS (or PIPES) 0.209g MOPS
    15% Glycerol 30ml 50% glycerol
    Sterile Water ??mL Water (to 100ml)
    • Adjust pH to 6.5 with KOH.
    • Filter w/ 0.45um and Store at -20C. (Pre-Rinse filter with sterile water)

    Transformation

    1. Thaw competent DH cells on ice for 10 minutes.
    2. Add DNA (not more than 10ul or 6 ng)
    3. Mix well and leave on ice for 15-45 min.
    4. Heat @ 42C for 90 sec.
    5. Put on ice for 1-2 min.
    6. Add 0.5 mL LB media.
    7. Incubate 1 hr @ 37C.
    8. Plate