Heat Shock Transformation of Chemically Competent Cells
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.
Incubator (or) Hot Water Bath, ideally shaking at 37°C
Heat Block or Hot Water Bath at 42°C
Foam floatie if using a water bath.
5-15 ml Sterile LB Liquid Media
Chemically competent cells of your desired strain (kept at -80°C)
Sterile LB Agar plates containing correct concentration of antibiotic corresponding to plasmid being inserted
Antibiotic stock solution corresponding to the plasmid being inserted
Ice box with ice
Protocol:
Day 1:
Remove one or more aliquots (as required) of chemically competent cells of your E.coli strain from the -80°C freezer. Thaw the cells e.g. by rubbing them in your hands or put them briefly in a 37°C waterbath, but don’t let them stay warm! As soon as they are thawed, put them onto ice.
Divide the cells into the appropriate number of 50 µl aliquots in separate Eppi tubes on ice. Add your DNA samples to each tube; you can use up to ~10 ul of ligation mixture or plasmid here, but note that typically 3 µl of ligation mix or 1 µl of plasmid would be standard.
Make sure you include both a positive control and a negative control in the transformation experiment. The positive control should be 1 µl of a plasmid with the correct antibiotic resistance (same resistance as the plasmid used for the ligation), and should also be a plasmid stock that you know is in good condition (based on agarose gel).The negative control is simply no DNA added.
Put the cells into a foam ‘floatie’ and put on ice. Ensure at least the bottom half of the tube (approx 2 cm) is embedded in the ice, don’t just rest them on top of the ice. Allow the cell/DNA mixtures to incubate on ice for 15-30 min.
Take your esky of ice over to the 42°C waterbath or 42°C heat block. Put tubes in a floatie (or) hold in the water bath (or) push tubes into the slots of the heat block. Allow 45 seconds for heat shock. (Plus or minus 10 seconds, this needs to be exact!). Then transfer the cells straight back onto ice (embed into ice, as above, don’t just rest on top).
Allow transformation mixtures to sit for 2 min on ice, then add 1 ml sterile LB broth to each tube. You can also use more fancy media (e.g. SOC or SOB), but there is not that much difference.
Incubate on 37°C shaker for 1 hour. Put the tubes horizontal so they get good shaking action. eg. put the tubes laying flat on the shaker platform and masking-tape into place. Make sure the lids are tight! You can incubate without shaking, and you can incubate for less time (30 min), but it won’t work as well in these cases.
The role of this ‘recovery’ step is to allow the cells to create the proteins required for antibiotic resistance.
Recovery is not necessary for Ampicillin resistance plasmids. Proceed to plating.
Label the LB-antibiotic plates before starting the next bit; you need two plates for each ligation condition or plasmid type, since we will plate out two different cell concentrations of each to ensure we get countable/pickable numbers of colonies. Double check the plates to ensure you are using the correct type of antibiotic(s) for the type of plasmid(s) you are using.
Pipette 100 µl of the first cell suspension onto one LB-antibiotic plate (label ‘100 µl’ in addition to other info) and spread it over the plate using the glass rod (liquid > solid) Sterile Technique.
Spread 100 µl of the remaining samples, each onto a separate, appropriately-labelled plate.
Centrifuge all the tubes at ~4,000 rpm for 1 minute in a micro-centrifuge. Pour off most of the supernatant into culture waste (being careful not to touch the tubes on the edge of the culture waste bottle). Leave a little bit of liquid behind (about one or two drops).
The purpose of this spin and second plating is to ensure you avoid getting too few colonies or a confluent lawn when you check your plates tomorrow.
Vortex the cells in the remaining liquid for about 10 seconds, until they are not sticking to tube anymore, and you have a nice smooth, even, cell suspension.
Pipette the cells from the first cell suspension onto the appropriate pre-labelled LB-antibiotic plate (label with ‘pellet’ in addition to other info), spread plate as above. Repeat for the remaining samples
Incubate all plates at 37°C overnight. Note that for some plasmids and ligations, it may be beneficial to instead try room temp for 2-3 days – this lowers the copy number of pUC type plasmids, and is useful to allow retrieval of clones that might be toxic to the host.
Day 2:
When examining your plates, first check your controls. The positive control should have thousands of colonies, perhaps even a confluent lawn of growth, especially on the ‘pellet’ plate. the negative control should have no colonies at all. If you don’t see these results with the controls, anything you see on your experimental plates is questionable.
Common problems and their interpretations:
Problem: No growth on your experimental plate or negative control, but plenty of growth on your positive control.
Your Heat Shock Protocol and Chemically Competent cell protocols are fine, something is wrong with the experimental plasmid.
Proceed to Ligation Troubleshooting
Problem: Lots of growth of the negative control (thousands of colonies or lawn). Interpretation:
Forgot to add antibiotic to the plates
Antibiotic concentration is wrong (too low)
Host bacteria are already resistant to the antibiotic (e.g. TOP10 has chromosomal streptomycin resistance)
Plates incubated too long (especially with LB-ampicillin)
Severe contamination with an antibiotic-resistant bacterium (not E.coli) (unlikely!)
Mix up of labelling somewhere – is this actually the positive control? or one of the experimental tests?
Problem: Some growth on the negative control (a few colonies). Interpretation:
Contamination during the procedure, e.g. from one of the other samples or the pipette etc. This may not be a ‘deal-breaker’ so long as there are lots more colonies on your experimental test plates
Antibiotic plates might be old, consider pouring fresh ones.
Mix up of labelling
No growth or very little growth on the positive control plate. Interpretation:
The cells are not competent
Used the wrong antibiotic in the agar (check the sequence of your plasmid to confirm correct resistance)
Used the wrong concentration of antibiotic (too much)
Agar plates are ‘bad’ for some other reason (e.g. added mercuric chloride instead of sodium chloride!)
Plasmid stock has gone bad (run a gel to check)
Mix up of labelling
Pipetting error (look at the pipette tip to ensure that you really have 1 µl of plasmid in there!)
Acknowledgements:
Coleman Protocols 2017 + 2019 http://coleman-lab.org/