for LB to be a plate, add 15g / liter order to LB broth
- 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
- The pre-labeled plastic petri dishes with antibiotics name, your name, and date
- 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) - swirl around solution
- Pour onto a plate
- when the plate solidify, somersaults
- Store in a cool room, with a plastic bag or wrap around spooky around it, and labeled with your name
transformation procedure
- take competent cells for disbursement (should only take 5-10 minutes) and start using it immediately afterwards
- + 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
- leave on ice for 30 minutes
- 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
- Also during this time, set the water bath to 42 degrees
- 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)
- put back on ice for 1-2 minutes
- add 100uL LB
- rocking at 37 degrees Celsius for anywhere from 20-45 minutes (usually about 40 minutes) in a block of metal
- 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 - The use of a long Pasteur pipette
- The thin-central fire until nearly melted, at which time, immediately move it out of the fire soon
- The angle of about 90 degrees should be established
- flame tip of a thin section for 5-10 seconds or more to prevent the entry of ethanol