Bacterial transformation
Chemically competent cells, usually made with cold calcium chloride, mixed with DNA on ice and then given 30–60 s at 42 °C. The standard method: cheap, needs no instrument, and good enough for almost all routine cloning at 10⁶–10⁹ cfu/µg depending on how the cells were made.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Heat shock
Chemically competent cells, usually made with cold calcium chloride, mixed with DNA on ice and then given 30–60 s at 42 °C. The standard method: cheap, needs no instrument, and good enough for almost all routine cloning at 10⁶–10⁹ cfu/µg depending on how the cells were made.
How it differs from the standard method
- Everything happens on ice until the heat shock; warming the cells beforehand costs ten-fold or more.
- Flick the tube to mix, never vortex: competent cells are fragile.
- 42 °C for 45 s is a compromise between uptake and survival. Minutes at 42 °C kills the culture.
- Tolerant of salt and of a few µL of ligation mix added straight to the cells.
1Getting a plasmid across the envelope
E. coli does not take up DNA on its own. You have to make it competent: either by soaking the cells in cold divalent cations, which neutralise the charge on the lipopolysaccharide and the DNA and leave the envelope in a fragile state that a brief jump to 42 °C pushes DNA through, or by washing all the salt out of them and driving the DNA in with a high-voltage pulse that punches transient holes in the membrane.
2Why cfu/µg only means something in the linear range
Efficiency is a ratio, and a ratio is only informative where the numerator rises in proportion to the denominator. With very little DNA, doubling the plasmid doubles the colonies, and cfu/µg is a constant that genuinely describes the batch of cells.
Linear
Colonies in proportion to DNA. This is where you measure a batch of competent cells, with a known supercoiled plasmid.
Half saturated
Still more colonies, but no longer proportionally. Quoting cfu/µg here understates the cells.
Saturated
Almost no extra colonies for a lot of extra DNA. Fine for a difficult ligation, meaningless as a measurement.
3The outgrowth, and why it matters more for some markers
After the DNA is in, the cells go into rich medium at 37 °C for twenty minutes to an hour before they are spread. This is not a recovery courtesy: the resistance gene has to be transcribed and translated before the cell meets the antibiotic.
4Reading the plate
A countable plate holds roughly 30 to 300 well-separated colonies. Below that the Poisson counting error gets large: a colony count is a count of independent rare events, so its relative standard error is about 1/√N, and a plate with 8 colonies carries about 35% error before you have made a single pipetting mistake.
- No-DNA control
- Competent cells carried through the whole procedure with no plasmid, spread on the same selective plate. It must be empty. If it is not, the plate is not selective (the antibiotic was left out, has been degraded, or the plate is old) and nothing on the sample plate can be attributed to transformation.
- Known-plasmid control
- A supercoiled plasmid you have used before, at a known small amount. It proves the cells were competent and the plating worked. When the sample plate is empty, this control is what separates a dead batch of cells from a problem with your own DNA.
- Vector-only control
- The ligation set up without any insert. If it gives as many colonies as the full ligation, both plates mean the same thing: the vector is closing on itself and the colonies on your ligation plate are mostly empty vector.
5Satellite colonies
On an ampicillin plate left too long at 37 °C, each resistant colony clears the drug in the agar around it, because β-lactamase is secreted and diffuses. Untransformed cells that were spread nearby then grow in that halo as a ring of tiny pinpricks.
Key terms
Practise these as flashcards →Bacteria in a treated state that lets DNA cross the envelope. The state is fragile: it decays above 0 °C and is largely destroyed by a freeze-thaw cycle.
Colony-forming units per microgram of DNA, measured with a known supercoiled plasmid at an amount small enough that colonies still rise in proportion to DNA.
The 20–60 min in rich medium at 37 °C between getting the DNA in and meeting the antibiotic, during which the resistance protein is made.
The decay time of an electroporation pulse, read off the instrument. A salt-free sample in a 1 mm cuvette gives about 5 ms; a salty one drops to well under 1 ms and usually arcs.
Cloning an insert into a reporter gene so that the gene stops working. In blue-white screening the reporter is lacZα and the colour is lost.
A tiny untransformed colony growing in the halo of cleared ampicillin around a resistant colony. Not a transformant, and not pickable.
When to use it
- Propagating a plasmid: putting a construct into E. coli so you can grow it up and prepare DNA from it.
- Recovering clones from a ligation or an assembly reaction, where the colonies you get are the candidates you will screen.
- Measuring a batch of competent cells you have just made, by transforming a known supercoiled plasmid at 0.1–1 ng.
- Getting an expression construct into a protein-expression strain, where the plasmid is already correct and all you need is a single colony.
Compared with alternatives
The step before this one: transformation is how you find out whether the ligation worked, which is why the vector-only control belongs on the same bench.
Overlap-based assembly makes circles without ligase, but they still have to be transformed, and assembly products transform worse than clean supercoiled plasmid.
What you do with the colony afterwards: grow it and prepare the DNA, then check it before you trust the clone.
The equivalent problem in eukaryotic cells, where the readout is expression in a population rather than colonies from single survivors.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Chemically competent cells, usually made with cold calcium chloride, mixed with DNA on ice and then given 30–60 s at 42 °C. The standard method: cheap, needs no instrument, and good enough for almost all routine cloning at 10⁶–10⁹ cfu/µg depending on how the cells were made.
- Everything happens on ice until the heat shock; warming the cells beforehand costs ten-fold or more.
- Flick the tube to mix, never vortex: competent cells are fragile.
- 42 °C for 45 s is a compromise between uptake and survival. Minutes at 42 °C kills the culture.
- Tolerant of salt and of a few µL of ligation mix added straight to the cells.