(last update : 18-10-1999)

CLONING


  1. Reagents needed :



  2. Preparation of competent E. coli cells :


    1. Inoculate 1 single colony of E. coli in 5 ml L-Broth medium and grow overnight at 37C in a shaking incubator.
      • (strain XL 1 Blue : add 3 l tetracycline to the 5 ml LB medium)
      • (strain DH5a : no additives in medium !!)
    2. Bring 20 l of this overnight culture in 100 ml LB medium. Grow the culture while shaking at 37C until OD550 = 0.3 to 0.5. (THIS OD IS CRITICAL !!!) (this takes 5 to 9 hours) (for faster growth : inoculate up to 1 ml culture in 100 ml medium)
      • (strain XL 1 Blue : add 60 l tetracycline to the LB medium)
      • (strain DH5a : no additives in medium !!)
    3. Cool the cultures on ice for 10 minutes. Collect the bacteria by centrifugation at 4.000 rpm for 10 minutes. Resuspend the pellet in 50 ml Ca/Mg solution. Incubate 30 minutes on ice.
    4. Collect the bacteria by centrifugation at 4.000 rpm for 10 minutes. Resuspend the pellet in 10 ml Ca/Mg solution. Incubate 15 minutes on ice.
    5. Add 3,4 ml sterile (60 %) glycerol to the 10 ml culture (endconcentration glycerol = 15 %) and resuspend. Devide the suspension into aliquots of 300 l (in 1,5 ml tubes). Store at -70C.

  3. Cleaning of PCR product :
  4. use High Pure PCR product purification kit (Boehringer).

    If the band on gel is weak, concentrate the product by using 50 l for cleaning on column and resuspend it in 20 l.

  5. Ligation :
  6. use pGEM-T and pGEM-T Easy Vector Systems (Promega).

    1. Set up ligation reactions as below :
    2. Mix and incubate overnight at 4C

  7. Transformation :


    1. Put a frozen tube with competent cells on ice until thawed
    2. Add 2 l of the ligate on the cells and mix gently
    3. Place on ice for 30 minutes
    4. Heat-shock the cells for 1,5 minute at EXACTLY 42C
    5. Put on ice for 2 minutes
    6. add 1 ml L-Broth medium to the tube and incubate 1 hour at 37C
    7. Spin the cells down and discard most of the supernatans : leave 100 l in the tube
    8. Prepare agar plates :
    9. bring 87 l Xgal (2 %) and 2 l IPTG (1M) on the plate and spread
    10. Suspend the cells, bring the 100 l suspension on the plate and spread
    11. Incubate overnight at 37C
    12. The white colonies are positive

    If you need to make plasmid DNA, perform 7. in stead of 6.

  8. PCR of the positive colonies :


    1. Pick a white colony with a toothpick and rub in in a PCR tube, repeat for several colonies
    2. Add PCR mixture to the tube and start the PCR
    3. Check the size of the PCR product on gel
    4. The fragments of the right size can be sequenced

  9. PCR of the positive colonies and plasmid isolation:
    1. Pick a white colony with a toothpick, rub in in a PCR tube and streak the cells also on a L-Broth agar plate (1 cm streak for each colony. When you mark the outside of the plate with a grid, you can inoculate up to 50 colonies on 1 plate). Repeat for several colonies. Incubate the plate at 37C.
    2. Add PCR mixture to the tube and start the PCR.
    3. Check the size of the PCR product on gel for the right fragments.
    4. During the PCR, the streaks from the positive colonies are grown. Inoculate the streaks, that gave an expected band in PCR, in 5 ml L-Broth and incubate at 37C overnight while shaking.
    5. Spin down the bacteria (2 times 1,5 ml) for 2 minutes at 6000 rpm.
    6. Prepare plasmide with the kit : High Pure Plasmid Isolation Kit from Boehringer.

back to homepage