Rapid* Transformation Protocol for Chemical Competent Cells
(*ONLY for AmpR Cells!)
- Place LB plates with appropriate antibiotic(s) at the appropriate temperature (37 °C typically, or only 30 °C for temperature sensitive expression systems).
- Thaw 25 ml of comp cells per plasmid or reaction (on ice, ~20 minutes). (Distribute, if necessary, 25 ml of comp cells to each pre-chilled transformation tube. Keep on ice.) DO NOT VORTEX or CENTRIFUGE Comp Cells!
- Dilute plasmid DNA to 2 to 50 ng/ml and add 1 ml to each tube containing comp cells on ice. (If using a ligation reaction, heat kill the ligase by incubating the reaction at 65 °C for 20 minutes (or 80 °C for 10 minutes) prior to transformation. Use 1 µl ligation reaction per transformation.)
- Wait 20 minutes, keep on ice.
- Heat shock tubes at 42 °C for 45 seconds. Place on ice immediately.
- Plate all of each transformation mix onto an appropriate pre-warmed LB + antibiotic plate (one transformation tube per plate) and incubate overnight at appropriate temperature (usually 37 °C but only 30 °C for temperature inducible systems).
For ligation reactions, always do a NO INSERT Control ligation ("or Minus Insert") as well as the actual recombinant reaction ("Plus Insert"), then transform 1 µl of each in separate transformation reactions, and plate these reactions as described above. Any "stimulation" in colony numbers due to the insert then will be immediately obvious and the number of colonies that must be preped to have a 99% probability of obtaining the correct recombinant can then be calculated according to:
N = ln(1-P) / ln(1-F)
Where
N = number of colonies that need to be picked and analyzed
P = probability expressed as decimal percent, eg. 99% would be 0.99
F = frequency calculated based upon the number of colonies on the plates as
F = (# on PLUS PLATE - # on MINUS PLATE) / (# on PLUS PLATE)
Eg. For a probability of 99% to get one correct recombinant colony where there are 15 colonies on the PLUS INSERT plate and 12 colonies on the MINUS INSERT plate, the equation would be:
N = ln(1-0.99) / ln(1-0.20) = -4.605 / -0.223 = 20.6 or 21 colonies !
This plot shows the curve for the number of colonies, N, that must be picked to have a 99% probability of finding one recombinant versus the frequency of colonies, F, on the PLUS INSERT versus the MINUS INSERT plates. Clearly, the number of colonies that must be picked climbs too high when the frequency drops below about 40%.
Protocol provided by Dr. John Brandis (02/24/2006) |