This protocol describes how to produce stocks of chemically competent DH5a E. Coli using calcium chloride and how to transform these cells using the heat-shock method. This is the most convenient method for making competent cells. It’s also the least rigorous and consequently transformation frequencies are relatively low, usually around 10^6 colonies per ug of circular DNA. One can obtain much higher frequencies (10^7-10^9 per ug) by using cells prepared by the Hanahan method or by electroporation. However, for most purposes, like introducing plasmid DNA into cells for stocks or most cloning applications, calcium-treated cells are fine. The higher transformation frequencies are usually needed only for introducing precious library DNA or recovering very rare clones in a mixture.
Preparing transformation competent DH5a E. Coli using CaCl2
- It is very important that sterile technique be strictly observed through the prep. All centrifuge bottles, tubes, solutions, pipets, etc. must be sterile. Any contamination during the prep will result in problems for everyone in the lab who uses the competent cells for cloning, thereby probably decreasing your popularity.
- Inoculate 1 ml of LB with cells from a frozen glycerol stock and incubate overnight at 37ºC.
- Transfer the entire 1 ml into a 1 L erlenmeyer flask containing 500 ml LB and incubate at 37ºC.
- Monitor growth starting at about 2 hours by measuring OD of an aliquot. Let cells grow until the OD550 = 0.45 to 0.6. This should take approximately 4 hours.
- Transfer the culture to 2 x 500 ml centrifuge bottles and chill on ice to 4ºC.
- Centrifuge at 6,000 g for 5 min at 4ºC.
- Pour off supernatant and pipette off remaining supernatant.
- Resuspend each pellet in 125 ml ice cold 50mM CaCl2. Combine into a single 500 ml centrifuge bottle.
- Centrifuge at 6,000 g for 5 min at 4ºC.
- Pour off supernatant and resuspend in 21.5 ml ice cold 50 mM CaCl2
- Add 3.5 ml sterile glycerol (100%) and mix gently
- Prepare 500 µl aliquots in sterile microfuge tubes and snap freeze in liquid nitrogen
- Store in -80ºC freezer. Use within 6 months if possible
Transforming chemically competent DH5a E. Coli using heat shock
- DH5α E. Coli cells are stored at -80ºc in aliquots of 500 µl. Thaw one vial on ice. Generally need 100 µl for each transformation. It is best not to refreeze whatever is leftover, just throw it away.
- For each transformation, pipet 100 µl of competent cells into a sterile 1.5 ml microfuge tube.
- Add DNA (5-10 ul of PCR product or 1 µl plasmid) to each tube and incubate on ice for 20-25 mins (can be as short as 5 min). This allows the DNA to adsorb to cell surfaces.
- Heat shock the cells/DNA to allow uptake of DNA into cells. Place tubes in 42º C water bath for 45 seconds. Variation: place all tubes at 370C for 2 mins. Return cells to ice.
- Add 1 ml of LB or SOC to each tube and incubate at 37ºC for 45-60 mins in a shaking incubator to let the cells recover from the heat shock and start growing again. Liquid should appear cloudy at this point.
- Streak the cells onto agar plates with the correct antibiotic. If you are using blue/white selection remember to coat the plates with 40 µl of 2% X-gal in DMSO at least one hour before streaking the bacteria. I usually streak two plates for each transformation. On one plate streak 50 µl of the 1 ml cell slurry. Spin the remaining 950 µl by placing in a table-top centrifuge and bringing the speed up to 12,000 g for one or two seconds. This should pellet the cells. Pour off the majority of LB/SOC, then resuspend the pellet in the remaining ~100 µl and spread this on the second plate.
- Incubate plates at 37ºC overnight.
- Pick colonies the next day