Competent cells and transformation controls

Competent cells are E. coli prepared to take up plasmid DNA. The preparation is either a chemical treatment, usually a calcium-class wash plus a heat shock, or electroporation, a high-voltage pulse across cells washed nearly free of salt. Choosing between them, and plating the controls that make the choice readable, is the decision this page supports. How a cloning strain is grown in the first place is in bacterial culture and transformation. What you do with a colony you trust is in plasmid cloning from insert to colony.

The photograph at the top is a Petri dish with blue and white bacterial colonies. It does not show a person. Blue and white are a colour screen, not proof that your cells, your drug or your ligation worked. Read the controls below before you read the colour.

Cell classes and related reagents can be specified from the molecular biology catalogue. Ratings on a label are colonies per microgram of a stated control plasmid. They are a class of performance to shop against, not a promise about your ligation.

Criteria you can apply before the cells thaw

Choose chemical competence when the DNA is a standard ligation or assembly in a salty buffer, the plasmid is an ordinary cloning size, and you need enough colonies to screen rather than a library. The method tolerates a small volume of ligation buffer because the cells are not waiting for a high-voltage pulse. It asks you for ice, a timed heat shock, and a recovery step without antibiotic.

Choose electroporation when the DNA is clean and low-salt, you have cuvettes and a pulser, and you need more colonies from less DNA. Large plasmids and rare assemblies are the usual reasons. The method fails closed when salt travels with the DNA: the pulse arcs, the cuvette cracks or pops, and that aliquot of cells is dead. Desalt, or dilute only within the limit the cell instructions allow. A Gibson mix or a ligation dropped straight into an electroporation cuvette is a salt problem until you have removed the salt.

Match the strain to the plasmid. A cloning strain used to build a T7 construct is not automatically an expression strain. Resistance already on the chromosome makes the antibiotic plate meaningless. Read the genotype before you buy a high-efficiency lot of the wrong host.

The three controls, and the number they are allowed to be

Plate a known circular plasmid of known mass on the same antibiotic, using the same cells and the same heat shock or pulse. Count colonies, correct for how much of the transformation you plated, and divide by the micrograms you added. That quotient is efficiency. It belongs to the control plasmid. Write the mass down at the time. A guessed mass is not a denominator.

Plate cells with no DNA on the same antibiotic. The plate should be empty at the density you can inspect. Colonies here cancel the other plates. They mean the drug never held, the cells were contaminated with a resistant strain, or you plated the wrong antibiotic for this marker.

Ligate or assemble the vector without insert, and transform that reaction in parallel with the real one. The colony count is self-ligation or self-assembly background. Your real plate means something only when it is clearly richer than this control, or when a later screen shows the insert in spite of a modest background. A no-insert plate as full as the test plate means you are about to screen parent vector. Stop and fix the join.

Recovery broth must not contain the antibiotic. The marker has to be expressed before the drug is fair. Then spread on selective agar. Plating the entire recovery onto one dish can exhaust the drug or produce a lawn of satellites on ampicillin. Plate a defined fraction so the controls are countable.

The Addgene bacterial transformation protocol shows how public notes lay out these controls. Times and voltages stay with the cells you thawed. protocols.io is a place those variants get written down. Neither is your pulse setting.

Heat shock timing and warm plates

Chemical competence depends on a cold incubation and a short heat pulse. Many laboratory strains are shocked near 42 Celsius. The number of seconds is a property of that cell protocol. Thirty seconds, forty-five seconds and longer pulses are all in use on different lots. Transfer the tubes when the bath is actually at the set point, start the clock then, and return them to ice as the protocol says. A pulse that runs long because the tubes were forgotten does not become a better efficiency. A bath that never reached the set point is a different reaction. Record the seconds you truly used.

Plates should be warm and dry enough that a 50 to 100 microlitre spread soaks in instead of beading. Cold plates from a refrigerator cut the colony count of the control plasmid and of the ligation together, which looks like incompetent cells. Wet plates smear colonies into one another so the no-DNA sector is no longer a clean control. Warm the plates at the incubation temperature with the lid handled so condensation does not drip back, and do not add a heat-labile antibiotic until the agar itself has cooled below the point that damages the drug.

Electroporation has its own timing. The pulse is milliseconds, set on the instrument for that cuvette gap. Recovery is still required before antibiotic. An arc is not a low-efficiency transformation you should plate anyway. Discard the arced cuvette, desalt the DNA, and repeat. Salt is what you are removing. More DNA in ligation buffer makes the arc more likely, not less.

How to reason when the plate is empty or carpeted

If the known plasmid gave no colonies, stop. The ligation cannot be interpreted. Check the antibiotic identity against the marker, the heat-shock clock, the expiry and storage of the cells, and whether the plates were cold. If the known plasmid grew and both ligations did not, the cells are fine and the join is not. If the no-DNA plate grew, none of the counts are efficiencies. If only the plus-insert plate grew, you still have candidates, not confirmed clones. Colony screening and insert orientation come after this gate. They do not replace it.

A carpet of growth is a lawn. It means selection did not happen. Do not pick "colonies" out of a lawn and call them transformants.

Safety

Treat the strain as your biosafety rules already classified it. The WHO Laboratory biosafety manual, 4th edition is background reading, not permission to choose a containment level. Non-pathogenic K-12 cloning strains are the context of this page. An insert that encodes a hazard does not become safe because the cells were competent. This is research practice, not a clinical susceptibility test.

Heat, ice and wet agar

In a hot room an ice bath becomes water while you label tubes, and a shallow beaker used as a 42 degree bath can run hotter than the thermometer at the edge. Hold competent cells on ice that still contains ice, and check the heat-shock bath in the liquid the tubes occupy, not in the air above it. On a humid day, agar poured and left covered sweats. Condensation on the lid drips across the no-DNA sector and drags cells with it. Dry the plates and the lids before you spread. Those two checks decide whether a poor colony count is the DNA or the room.

What to put in an enquiry

State chemical competence or electroporation, the strain genotype, the antibiotic marker, the approximate plasmid size, and whether the DNA will be a salty ligation or a desalted sample. Ask for the control-plasmid rating as a class, then plan to measure your own. A construct someone else will build can be discussed through the custom gene synthesis enquiry reference. A later sequence check can be framed with the Sanger sequencing enquiry reference. Neither page means EVRINTH makes the cells competent, synthesises the gene or runs sequencing. Send the requirement with the quote request and ask whether a quotation is possible.