Transformation of Competent Cells Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Transformation of Competent Cells Protocol

for LB agar plates, add 15g/litre of Agar to the LB broth
1. using the liquid cycle, autoclave the above recipe in a sufficiently large bottle (the solution should use only 80% of the maximum volume of the bottle), remembering to loosen the cap
2. pre-label plastic petri plates with the antibiotic name, your name, and the date
3. when the solution is cool enough that the hand can hold it, add antibiotics:
ampicillin—add 1ul per ml of LB agar (stock concentration of ampicillin at 100 mg/ml; final concentration ampicillin at 100 ug/ml; stock concentration of ampicillin made in sterile water, mixed, filtered, and stored in 1 ml aliquots at - 20 degrees C)
4. swirl around the solution
5. pour onto the plates
6. when plates solidify, turn upside down
7. store in the cold room, with either a plastic bag around it or seram wrap around it, and labelled with your name

Transformation procedure
1. take out competent cells to thaw (should only take 5-10 minutes) and start to use it soon afterwards
2. + 1.25uL of PCR plasmid (e.g. from site-directed mutagenesis) or 5uL of ligation reaction (half the amount of the ligation) to 50uL of cells; always include a negative control transformation: for PCRs, transform a PCR that had one of the primers missing; for ligations, transform the digested vector only
3. leave on ice for 30 minutes
4. during this time, put agar plates from cold room into the 37-degree incubator, with the cover open and the plate turned upside down
5. also during this time, set the water bath to 42 degrees
6. one 30 minutes is up, heat shock the bacteria in the 42-degree water bath for 60 seconds—time it exactly; between 45-90 seconds is OK (if using the metal heating block, it may be necessary to do the heat shock for 90 seconds, but don’t press the eppendorf tubes down too low, or they’ll be hard to take out)
7. put back on ice for 1-2 minutes
8. add 100uL LB
9. shake at 37 degrees Celsius for anywhere from 20-45 minutes (usually about 40 minutes) on the metal block
10. plate the cells on agar plates by pipeting the cells onto the middle of the plate and spreading: before use of the spreader each time,
dip the spreader in ethanol and flame the spreader, let it sit for 1 minutes before spreading, and then touch the spreader onto an area of the agar plate without cells so that the spreader is not to hot before spreading
How to make a glass spreader for use in transformations
1. use a long Pasteur pipet
2. flame the middle of the thin section until it’s almost about to melt, upon which time, immediately move it away from the flame immediately
3. an approximately 90-degree angle should have formed
4. flame the tip of the thin section for 5-10 seconds or so to prevent ethanol from entering it