1、精選文檔Electrocompetent Cell Preparation1. Streak out bacterial strain to be used on LM plates (not LB). Restreak (without cooling) a single colony again on LM the next day to further increase competency.2. Prepare several liters of ddH20, and 100 ml of 10% glycerol ahead of time and store in cold room

2、 the day before making competent cells. Do not autoclave glycerol, filter through 0.45 mm filter.3. Pick 10 fresh, juicy, actively growing bacterial colonies and culture them in 100 ml pre-warmed SOB in 1000 ml flask, shaking vigorously at 37°C (250 rpm OK). This takes 1-3 hours. (See alternate

3、 step 3A below).4. When culture beginning to get cloudy (but O.D.600 < 0.25), split into four 250 ml cultures of prewarmed SOB, for final volume of 1.0 liter. Use four 2.0 liter flasks. Continue to shake vigorously at 37°C. Periodically check O.D.600 . (See alternate step 4A below).5. When O

4、.D.600 0.50-0.60, collect cultures. This will take 1-3 more hours. Collect into chilled GS-3 rotor tubes. These hold about 500 ml each. Perform all subsequent steps at 4°C or on ice until freezing of cells. 6. Spin 4,000 rpm at 4°C for 20 minutes. Most strains will pellet at this speed - b

5、ut some dont. If SN is cloudy, try 5,000 rpm. Note that as the cells become more thoroughly washed, higher speed spins are necessary.7. Resuspend pellets in 500 ml cold ddH20. Pellet again in GS-3 at 5,000 rpm, 4°C, 20 minutes.8. Resuspend pellets in 500 ml cold ddH20. Pellet again in GS-3 at 6

6、,000 rpm, 4°C, 20 minutes.9. Resuspend pellets in 25 ml 10% glycerol in ddH20. Filter glycerol solution through 0.45 mm filter before using. Do not autoclave glycerol. Use 50 ml disposable centrifuge tubes and spin 6,500 rpm, at 4°C, 25 minutes, in HS-4 swinging bucket rotor. Use one tube

7、for cells and a balance.10. Resuspend cell pellet in 3.0 mls sterile 10% glycerol solution. Place 200 ml aliquots into microfuge tubes, cap them tightly. Have tubes pre-labelled with strain name, and notebook page number.11. Place directly in -80°C freezer. Alternate Approach with Overnight Cul

8、ture: Substitute steps 3A-4A for steps 3-4 above.3A. Pick one fresh colony from LM plate (never cooled), culture overnight in 100 ml pre-warmed SOB in 1000 ml flask, shaking vigorously at 37°C (250 rpm OK). Start this overnight culture as late as possible before going home. 4A. The next day, fi

9、rst thing in the A.M., inoclulate four 250 ml cultures of pre-warmed SOB with 0.5 ml each of the fresh overnight culture. Use four 2.0 liter flasks. Shake vigorously at 37°C (250 rpm OK). Periodically check O.D.600 . Proceed to step 5 above. This culture step will take 3-5 hours.LB MEDIUMfor 1

10、Liter=10 g bacto-tryptone 5 g bacto-yeast extract10 g NaClAdjust pH to 7.0 with 5 N NaOH (about 0.2 ml). Autoclave.Lab distilled water OKLM PlatesThes plates are for culturing bacteria to be used for making competent cells or electrocompetent cells.for 1 liter=10 g bacto-tryptone5 g yeast extract0.5

11、8 g NaCl15 g Bacto AgarAdd 800 ml tap H20pH to 7.0Adjust to 1.0 liter with tap H20AutoclaveAfter autoclaving, when cooling, add 10 mls 1.0M MgSO4 Note - these plates dont work with tetracycline since they contain magnesium.SOB MEDIUMfor 1 liter=20 g bacto-tryptone5 g bacto-yeast extract0.5 g NaClDis

12、olve in 950 ml waterMake 250 mM KCl:1.86 g disolved in 100 ml waterMake 5 N NaOH:2 g for 10 mlMake 2 M MgCl2:Disolve 19 g in 90 ml water. Bring up to 100 ml. Autoclave.Add 10 ml of 250 mM KClAdjust pH to 7.0 with 5 N NaOH (about 0.2 ml)Bring volume up to 1 literAutoclaveSOC MEDIUMfor 1 literThe same as SOB medium with the addition of 20 mM glucose.Make 1 M glucose:18 g in 90 ml of waterBri