Transformation of Competent Cells Protocol

for LB to be a plate, add 15g / liter order to LB broth

  1. The use of liquid cycles, autoclave recipe above in the bottle big enough (the solution should be to use only 80% of the maximum volume of the bottle), remember to loosen the lid
  2. The pre-labeled plastic petri dishes with antibiotics name, your name, and date
  3. when the solution is cold enough that the hand can hold, add the antibiotics:
    ampicillinadd 1UL per ml LB agar (stock concentration of ampicillin at 100 mg / ml; the final concentration of ampicillin at 100 ug / ml ampicillin stock concentration was made in sterile water, mix, filtered, and stored in 1 ml aliquots at – 20 degrees C)
  4. swirl around solution
  5. Pour onto a plate
  6. when the plate solidify, somersaults
  7. Store in a cool room, with a plastic bag or wrap around spooky around it, and labeled with your name

transformation procedure

  1. take competent cells for disbursement (should only take 5-10 minutes) and start using it immediately afterwards
  2. + 1.25uL of PCR plasmid (eg from site-directed mutagenesis) or 5uL ligation reaction (half the number of ligation) to 50uL cells; always include a negative control transformation: to PCR, PCR has changed the primary one is lost; for ligations, change the vector digested only
  3. leave on ice for 30 minutes
  4. during this time, put the plate in order from a cold room into an incubator 37 degrees, with the cover open and the plate inverted
  5. Also during this time, set the water bath to 42 degrees
  6. the 30 minutes are up, the heat shock bacteria in bath water of 42 degrees for 60 secondstime exactly; between 45-90 seconds OK (if using a metal heating block, it may be necessary to heat shock for 90 seconds, but do not press down too low eppendorf tube, or theyll hard to take)
  7. put back on ice for 1-2 minutes
  8. add 100uL LB
  9. rocking at 37 degrees Celsius for anywhere from 20-45 minutes (usually about 40 minutes) in a block of metal
  10. The cell plate on agar plates with cell pipeting into the middle of the plate and spread: before using the spreader each time,
    dipped in ethanol and flame spreader spreader, let it sit for 1 minute before it spreads, then touch the spreader into the agar plate without a cell area so that no heat spreader before it spreads
    How to make a glass spreader to be used in transformation
  11. The use of a long Pasteur pipette
  12. The thin-central fire until nearly melted, at which time, immediately move it out of the fire soon
  13. The angle of about 90 degrees should be established
  14. flame tip of a thin section for 5-10 seconds or more to prevent the entry of ethanol