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Preparation of Electro-competent E.coli- TG1

  1. Inoculate 15 mL of pre-warmed SB in a 50 mL flask with a single E.coli. Colony from a glycerol stock that has been freshly streaked onto an agar plate. Grow overnight at 250 rpm and 37oC.
  2. Dilute 2.5 mL of the culture into each of six 2 liters flasks with 500 mL of SB. Shake at 250 rpm and 37oC until the optical density (OD) at 600 nm is about 0.7.
  3. Pour the flask cultures into six pre-chilled 500 mL centrifuge bottles and spin at 3000 g for 20min at 4oC.
  4. Pour off the supernatant and re-suspend each of the pellets in 25 mL of pre-chilled 10% glycerol using 25 mL pre-chilled plastic pipette. Combine two re-suspended pellets in one pre-chilled 500 mL centrifuge bottle and add pre-chilled 10% glycerol up to about 500 mL. Combine the other pellets similarly. Spin as before.
  5. Re-suspend each pellet in 500 mL of 10% pre-chilled glycerol. Spin as before.
  6. Pour off the supernatant and re-suspend each pellet in 25 mL of pre-chilled 10% glycerol until complete homogeneity is reached. Transfer the suspensions into pre-chilled 50 mL tubes and spin at 2500 g for 15 min at 4oC. Meanwhile, set up about 50 1.5mL micro-centrifuge tubes in a rack in a dry ice/ethanol bath.
  7. Carefully pour off the supernatant from each tube until the pellet begins to slide out. Discard the supernatant. Using a 25 mL pre-chilled plastic pipette, re-suspend each pellet in the remaining volume and combine the 3 suspensions. Use a 1 mL pipet tip with a snipped–off end to immediately aliquot 300 mL volumes into the micro-centrifuge tubes that were placed in the dry ice/ethanol bath. Cap the tubes and store them at -80oC.

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