Rubidium Chloride Preparation of Chemically Competent E.coli Cells for Heat Shock Transformation
(Gold Standard, more expensive and dangerous)
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 Liquid 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 or ideally a full cold room.
RF1 Solution
200 ml dH2O
2.4 g RbCl (100mM)
2.0 g MnCl2 (50mM)
0.6 g K Acetate (30mM)
0.3 g CaCl2 (10mM)
30 g Glycerol (15% w/v)
Adjust pH to 5.8 with conc. acetic acid. pH will change very quickly and only requires ~ 10µL of acetic acid. Sterilize by filtration into an autoclaved media bottle.
RF2 Solution
100 ml dH2O
0.2 g MOPS (10 mM)
0.1 g RbCl (10 mM)
1.0 g CaCl2 (75 mM)
15 g Glycerol (15% w/v)
Adjust pH to 6.8 with NaOH or HCl, as appropriate. Sterilize by filtration into an autoclaved media bottle.
Protocol:
Day 1:
Set up a sterile workspace, ideally in a laminar flow hood. Wipe everything (including your hands/gloves) down with 70% ethanol.
Using sterile technique streak E. coli strain (e.g. JM109, TOP10, DH5a) from glycerol stock or other source onto plain LB agar (no antibiotics). Incubate overnight at 37°C.
Day 2:
Check that the plate contains homogeneous, pure colonies. Pick up a few colonies using sterile technique and then inoculate a 5mL plain LB broth with from the LB plate.
Incubate overnight (16-24 h) at 37°C, with shaking.
Day 3:
Aseptically inoculate 100mL plain LB broth (in a 500mL Schott bottle or Erlenmeyer flask) with 3 ml of overnight culture; this should give an initial OD600 of ~0.05. (check this to be sure)
Be sure to zero the spectrophotometer with sterile blank LB liquid medium.
Grow cells at 37°C with shaking, until the culture reaches OD600 of ~0.5 (anywhere from 0.3-0.7 is OK, but try to get close to 0.5). This should take 2-3 hours, so check the OD600 every 15 minutes or so after the first hour of incubation.
Aseptically inoculate 100mL plain LB broth (in a 500mL Schott bottle or Erlenmeyer flask) with 3 ml of overnight culture; this should give an initial OD600 of ~0.05. (check this to be sure)
Grow cells at 37°C with shaking, until the culture reaches OD600 of ~0.5 (anywhere from 0.3-0.7 is OK, but try to get close to 0.5). This should take 2-3 hours, so check the OD600 every 15 minutes or so after the first hour of incubation.
Aseptically transfer (pour) the cells into sterile centrifuge tubes or bottles (one 250 ml Sorvall bottle or 2 x 50 ml Falcon tubes) and balance these, either by transferring culture aseptically between the tubes/bottles or by adding sterile water or LB to the lighter tube/bottle.
Centrifuge at 4000 rpm (~3000 g in Centaur/Centurion machine) at 4°C for 10 minutes. Note: centrifuge needs to be cold. Turn on and set temperature beforehand. You can spin faster, up to say 7000 g, but above this point, faster is not better, and cell pellets will be hard to resuspend.
Working in the cold room with the cells on ice, pour off the supernatant into culture waste (don’t let the centrifuge tube/bottle actually touch the edge of the culture waste bottle). Try to remove as much of the liquid as possible – give it a shake / tap to assist this.
Resuspend the pellet gently in 33mL RF1 solution by vortexing and/or shaking the tube/bottle. Its OK to be rough with the cells at this stage in the process, but you shouldn’t need to shake or vortex for more than 10 sec or so.
Incubate on ice for 1 hour, then pellet the bacteria again at 4000rpm, 4°C for 10 minutes.
Working in the cold room, pour off the supernatant into culture waste. As before, try to remove as much of the residual liquid as possible.
Resuspend the pellet in 8mL RF2 solution by vortexing or shaking. At this stage, the cells have become more fragile due to the RF1 treatment, so its important not to shake/vortex any longer than ~10 sec (this shouldn’t be necessary).
Incubate on ice for 15 minutes. While the cells are incubating, set up all your Eppi tubes (~50) on ice with the lids open, so they are pre-chilled, and ready to receive cells. Label these tubes on top with the strain name before putting them on ice (labelling becomes difficult with cold and wet tubes!). Be careful to only push the tubes only about 2/3 of the way into the ice. If they are pushed in too far (right up to the lip of the tube), you risk getting ice or melted ice (not sterile!) into your cell aliquots
Working quickly (but still carefully!), aliquot 220 µL of cell suspension into the pre-chilled Eppi tubes. Once dispensed, close tube lids tightly, and collect all tubes into a bag/box with a clear and prominent label and store it immediately in the ‑80°C freezer. It’s a good idea to label both the outside of the bag/box AND place a label written on paper inside.
Test the transformation efficiency of the freshly-prepared competent cells using a known amount of a plasmid standard (see protocol for heat shock transformation).
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:
Coleman Protocols 2017 + 2019 http://coleman-lab.org/