Preparation of Electro-competent E.coli- TG1
- 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.
- 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.
- Pour the flask cultures into six pre-chilled 500 mL centrifuge bottles and spin at 3000 g for 20min at 4oC.
- 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.
- Re-suspend each pellet in 500 mL of 10% pre-chilled glycerol. Spin as before.
- 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.
- 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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