Making ultra-competent E.coli for heat-shock transformation (Inoue method)
Within this protocol:
Making ultra-competent cells
Making Inoue buffer
Making SOB and SOC
Transforming the cells
Calculating transformation efficiency
To make ultra-competent cells:
- Start a culture from a plate - from a single colony, inoculate and grow a 25mL LB culture in a 125mL flask at 240rpm 37°C. They recommend growing this 6-8h during the day then inoculating the next cultures at 6pm that evening.
- From this first culture, inoculate three 1L flasks with 250mL SOB and 2mL, 4mL, and 10mL of starter culture - grow at 18°C O/N shaking at 150-180rpm. Start all 3 of these so at least one is the right concentration (OD 0.55) in the morning (~18h)! NOTE: this growth can be really variable, taking even 24h+ growing at 18-20°C to get to OD 0.5
- Grow the culture to OD 0.55
- Ice the cell culture 10 mins to stop the growth
- Spin the cells (using the Theg lab centrifuge) in 50mL Falcon tubes at 3250rpm, 10min, 4°C. NOTE: spinning the cells in 50mL tubes gives just a small pellet in the very bottom of the tube
- Carefully pour off the supernatant fluid
- Resuspend the cells in 16mL ice-cold Inoue buffer per 50mL tube
- Ice the resuspended cells 10 mins
- Spin the cells again at 3250rpm, 10min, 4°C
- Carefully pour off the supernatant fluid
- Resuspend the cells in 4mL ice-cold Inoue buffer per 50mL tube
- add 300μL DMSO per 50mL tube
- Ice the cells 10 mins. While the cells are chilling, prepare about 80 eppendorf tubes for aliquoting, and prepare a container with liquid nitrogen for flash-freezing the aliquoted cells
- Aliquot 100μL cells per eppendorf tube
- Freeze the cells in liquid nitrogen
- Store at -80°C
To make Inoue buffer:
- Make a 50mL filter-sterilized 0.5M PIPES solution (pH 6.7) and freeze it at -20°C in 5mL aliquots.
For 100mL Inoue buffer (more than enough for 100mL cells),
- Add 1.08g MnCl2·4H2O to a final concentration of 55mM
- Add 0.22g CaCl2·2H2O to a final concentration of 15mM
- Add 1.86g KCl to a final concentration of 250mM
- Add 2mL 0.5M PIPES to a final concentration of 10mM
- Filter sterilize this solution for use in the competent cell protocol. NOTE: this can be stored at -20°C.
- NOTE: This can be made by adding PIPES salt directly to the solution, but if so wait to add the MnCl2·4H2O until after pHing to 6.7, then filter sterilize.
To make 1L SOB media:
- Add 20g tryptone
- Add 5g yeast extract
- Add 0.5g NaCl
- Add 10mL 250mM KCl (autoclaved)
- pH to 7.0 with NaOH
- Distribute this to smaller-volume bottles before autoclaving
- Before use, add 5mL/L 2M MgCl2 to the media.
To make SOC from SOB:
- Add 5mL 1M glucose (filter sterilized) to 250mL SOB
- SOC will become contaminated easily so it's best to store in small volumes
To transform with these cells:
- Use 50μL cells per transformation
- Thaw the cells on ice
- Add 0.5-1μg DNA or ligation to the cells
- Incubate on ice 30 min (at least 10 min, but longer is better)
- Heat shock 42°C for 45sec
- Incubate on ice 2 min (this step is important)
- Add 600uL SOC for recovery (SOB or LB can also be used)
- Shake cells at 37°C for 1h
- Plate 10-100uL cells on selective media
- Grow up O/N at 37°C
To calculate transformation efficiency:
- For each batch of competent cells you make, it's a good idea to calculate transformation efficiency.
- Transform the cells with a known amount of DNA (e.g. 1μg DNA into 100μL cells).
- Plate several volumes of cells onto selective plates (10μL, 50μL, 100μL cells per plate).
Transformation efficiency = # colonies/amount of DNA on the plate (μg/mL)
e.g. you count 1500 colonies on a plate
100μL cells
+ 1μL DNA @ 1μg/mL
+600μL SOC
711μL → plated 10μL
10/711 = 0.014 = "amount of DNA on the plate"
1500 colonies / 0.014 = ~1x105
Ideally, "ultra-competent cells" will have a competency of about 107 or 108.
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