Calcium Chloride Preparation of Chemically Competent E.coli Cells for Heat Shock Transformation
(safer but less efficient than RbCl)
Equipment and Consumables:
Sterile workspace (very important for this experiment)
Ideally perform within a laminar flow hood. Under a flame is fine if you have excellent technique.
Refrigerated Centrifuge capable of holding 50 ml tubes.
Putting a non-refrigerated centrifuge in a bar fridge is a commendable work around.
Spectrophotometer capable of measuring cell density
Note to self: Post ways to DIY this. They do exist.
Cuvettes for spectrophotometer
Vortex
Incubator (or) Hot Water Bath, ideally shaking
100-200 ml sterile LB medium in bottle with lid or foil cap
5-15 ml Sterile LB Liquid Media for overnight culture in smaller tube
LB Agar plate with a recently grown colony of the strain you plan to make competent (e.g. DH5α, BL21, etc.)
If the plate is old, take an extra day to replate the bacteria onto fresh agar to restart their growth cycle.
Antibiotic Stock Solution (if you’re trying to insert a second plasmid into a plasmid-containing bacteria)
Most of the time you will do this protocol with no antibiotic
Ice box with ice
25 ml of 0.1 M MgCl2 Solution (filter sterilised or autoclaved) kept on ice during experiment
25 ml of 0.1 M CaCl2 Solution (filter sterilised or autoclaved) kept on ice during experiment
2.5 ml of Cryo Buffer: 1.7 ml 0.1 M CaCL2, 0.3 ml 100% Glycerol (filter sterilised or autoclaved) kept on ice during experiment
Protocol:
Day 1:
Set up a sterile workspace, ideally in a laminar flow hood. Wipe everything (including your hands/gloves) down with 70% ethanol
The cells you produce during this experiment will have an effect on many of your downstream protocols. Taking extra care here to use good sterile technique will save a lot of tears down the road.
This is a relatively safe experiment, PPE is less necessary and may pose a contamination risk. Use at your own discretion.
Inoculate a single colony of appropriate cells from your plate using sterile technique and add them into your culture tube containing 5-15 ml Sterile LB liquid media. Add antibiotic if needed and culture overnight at 37°C, 200rpm.
Day 2:
Inoculate 100 ml LB with 1 ml of your o/n culture.
Gently swirl to mix the culture and then use sterile technique to pipette 1 ml into a cuvette labelled “0”.
Use sterile LB media to zero the spectrophotometer. Measure OD600 the T = 0 samples, it should be ~0.05
Incubate your 100ml culture at 37 °C, with 200 rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
If you don’t have a shaking incubator, consider inoculating with 2-3 ml of your o/n culture, otherwise you might be here for 5 hours.
The spectrophotometer should be set to measure the optical density at 600 nm. Look up a youtube guide based on your specific brand of machine. Or make your own DIY!
Take a sample after 90 minutes to check how much the cells have grown. Then another every 20-30 minutes. They will start slowly, taking two hours to go from 0-0.2 and then ZOOM from 0.4->0.7 without you noticing. This makes perfect sense since you’re trying to catch them right at the start of their ‘exponential phase’ of growth.
Transfer your cells to 2 x 50 ml sterile Falcon tubes using sterile technique and leave on ice for 30 mins.
Centrifuge at 400 x g, 5mins, 4°C.
Pour off the supernatant into culture waste.
Add 25 ml ice cold 0.1 M MgCl2.
Gently resuspend the cells in each tube using the vortex.
Incubate the Falcon tube on ice for 30 min.
Submerge the tube until the liquid is below the ice level, but not the lid. The ice is not sterile!
Centrifuge at 4000 x g, 5 min, 4 °C.
Pour off the supernatant into culture waste
Add 25 ml ice cold 0.1 M CaCl2.
Gently resuspend the cells in each tube using the vortex.
Incubate the Falcon tubes on ice for 30 min.
While waiting, set up 25 sterile microcentrifuge tubes on ice.
You can choose to set up more tubes if you need to scale up the protocol. You can do that, just multiply all the values by the same number!
Centrifuge at 4000 xg, 5 min, 4 °C
Pour off the supernatant into culture waste.
Add 1.25 ml ice cold sterile Cryo Buffer (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
Resuspend the cells in the Cryo Buffer very gently using the vortex.
Aliquot 100 µl of cell suspension into each microcentrifuge tube on ice, sealing each as you go.
Store the tubes in a labelled bag at – 80 °C
Streak out a loopful of the cells onto plain LB agar (using either a sample of a frozen aliquot or some residual cells remaining in the large centrifuge tube/bottle), and incubate at 30°C for three days to allow any of the common types of contaminants (e.g. Staphylococcus) to grow. This is to check the purity of the cell stock. The streak-plate should look completely uniform, with colonies of only one type (E.coli), and no heterogeneity in the initial patch or the streaklines which would indicate a mixture of bacteria is present.
When using cells for heat shock, only remove one vial from the freezer at a time. Repeated freeze/thaw cycles will destroy all of the cells.
Acknowledgements:
2017 Edinburgh iGEM Team Heat Shock Protocol: http://2017.igem.org/wiki/images/3/3b/T--Edinburgh_OG--chemcompcells.pdf