Competent Cells

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Prepare the following media and buffers (contents found in recipe book):

• LB +10mM MgCl2 plates ~ 100ml volume is enough
• 50ml TYM
• 950ml TYM
• 400ml TfbI
• 40ml TfbII
• 200 sterile eppendorf tubes
• autoclave two 1L plastic bottles
• Liquid N2

• Streak bacteria from original stock on LB +10mM MgCl2 (add CAM for BL21) and incubate overnight @37oC
• Inoculate a 50ml broth of TYM with 1 colony and shake @200rpm at 37oC overnight
• Take 1ml from the 950ml broth to use as a blank for the spectrophotometer.
• Pour "in a sterile manner" the 50ml culture into the 950ml broth and shake @200rpm @37oC until an O.D. @595nm of 0.9 is reached (note that doubling time for E.coli is ~20min)
• Pour the cells in 2 sterile plastic bottles and spin using the sorval @3000rpm for 15min @4oC
• Discard the supernatant and keep the cells on ice
• Resuspend in 400ml of TfbI
• Leave on ice for 20min
• Resuspend GENTLY with 40ml TfbII
• Aliquot the cells in the cold room: 200ul per eppendorf tube
• Immerse the cells in liquid nitrogen
• Store the cells in the cold room