Heat shock and electroporation concepts

Heat shock and electroporation are two different ways to get plasmid DNA into a non-pathogenic E. coli cloning strain, and they fail for different physical reasons. The decision this page supports is which failure you are looking at when the antibiotic plate is empty. The shared culture frame is bacterial culture and transformation. The DNA that should have circularised is plasmid cloning from insert to colony. The plate you spread onto is only meaningful if the drug matches the marker, which is the point of antibiotic selection and blue-white screening.

Cell preparations, cuvettes and recovery media are classes in the reagents and chemicals catalogue, read beside the molecular biology pathway. If the construct itself is under discussion, use the custom gene synthesis reference as an enquiry prompt and the quote request for the specification. A method can be discussed. This page does not set instrument programmes.

Sort the pulse before you blame the plasmid

A blank plate has a long list of causes. The useful first cut is the method you actually ran. Chemical competent cells expect a cold incubation, a brief heat pulse, ice again, and recovery. Electrocompetent cells expect a salt-free, ice-cold suspension in a cuvette, an electrical pulse defined by that cuvette and that instrument, and immediate recovery medium. Mixing the two sheets is a reliable way to kill both.

Keep a known supercoiled plasmid, dissolved in water, as the splitter. Run it beside the experimental DNA in the same cell lot, with the same pulse. If the known plasmid forms colonies and the experimental DNA does not, the pulse hardware and the cells are innocent enough. Look at the new DNA: it may be absent, linear, or loaded with ligation salt. If the known plasmid also fails, the cells or the pulse or the plates are the story, and redesigning the insert will not help.

The Addgene bacterial transformation protocol shows how a public cloning resource separates these methods. Follow the sheet that belongs to your tubes. NIST is the metrology reference for why a displayed temperature or a displayed voltage still needs a check against the instrument you are standing in front of. A set point is not a measurement.

What the heat pulse is doing

Chemical competence leaves the cell envelope able to admit DNA when it is cold and then briefly warmed. For a great many E. coli K-12 preparations that warm step is near 42 Celsius and well under a minute. Some lots specify a shorter shock, some a slightly different temperature, and some chemical methods rely on a cold incubation without a dramatic shock. The exact seconds are the manufacturer's, or the recipe you used when you made the cells. Copying "45 seconds" from a paper that used a different buffer is how viability collapses while the notebook still looks obedient.

After the pulse the tube returns to ice. Leaving it in the block while you find a pipette is an unplanned extension of the shock. Cells die, and the death looks like a cloning failure. Equally, skipping the shock on a method that requires it leaves the DNA outside. You also get no colonies. Those two mistakes are opposites, and the notebook should say which one you are at risk of. Measure the block. A display that reads 42 can hide a well at 37 or a well at 47. Use a thermometer in a spare well with a little water, once, when you set the instrument up.

Heat shock does not forgive a huge volume of cold ligase buffer, but it is more tolerant of residual salt than a cuvette is. A ligation that fails by arcing in an electroporator may still be worth a chemical transformation after you reduce the buffer load. That is a method choice, not a promise.

What the electrical pulse is doing

Electroporation drives DNA across the envelope with a short electric field. The cells are washed into a low-ionic-strength buffer and kept ice-cold. The cuvette gap and the instrument define the pulse. This article will not quote voltages, resistances or time constants, because those numbers are a programme for a particular gap and a particular machine. Follow the cuvette maker and the instrument maker together. A 1 millimetre gap is not a 2 millimetre gap. Using the wrong programme is how you either under-pulse or explode the suspension.

Arcing is the signature failure. You may hear it, see a flash, or read an error. Salt conducts. Ligation buffer, residual medium, and even a wet streak on the outside of the cuvette can provide the path. Bubbles in the gap do the same. The cells from an arced pulse are not a transformation to be plated "just in case" as if nothing happened. Prepare the DNA again in water, chill a dry cuvette, and use a fresh aliquot.

Recovery medium is added at once, often before the cells have time to sit in the low-salt buffer without nutrients. Delay here costs viability. Then, as with heat shock, you wait without antibiotic so the marker can be expressed. Spread a fraction. Electroporation of a clean supercoiled plasmid is often efficient enough that plating the entire cuvette gives a lawn of true transformants you cannot pick. Plate less when the DNA is a pure plasmid, and more when it is a rare assembly. That judgement is about colony separation, not about hiding a failure.

Symptoms, and which method they belong to

Empty plates that are not a pulse problem

Recovery omitted, plates poured so hot that the antibiotic died, plates dried to a crisp, or an incubator left at the wrong temperature will empty a plate after a perfect pulse. So will the wrong drug. A bla plasmid on kanamycin is not a heat-shock mystery. Work those down with the no-DNA plate and the known plasmid before you open the electroporator manual.

A lawn on every plate, including no DNA, is the opposite fault: selection never happened. Do not tighten the pulse to "reduce colonies". Add the antibiotic story back. Tiny satellites around a few large colonies are an ampicillin-halo problem, not evidence that the field strength was clever.

If both the known plasmid and a carefully desalted experimental DNA fail only in the cuvette, and the same cells heat-shock successfully, the instrument or the cuvette lot is the remaining branch. Try a second cuvette gap only if the manual offers a matched programme. Do not invent voltages.

Safety of a block and of a pulse generator

Both methods use a non-pathogenic cloning strain under the containment your institution has already set. The new hazards are physical. A heat block burns. An electroporator stores a pulse that must stay inside the cuvette holder. Do not defeat interlocks. Do not touch the electrodes. Solvents used to clean DNA and the antibiotics on the plates have their own safety notes. This page is not medical advice and not a procedure for introducing DNA into a pathogen. If the organism is not a cloning strain on your institutional list, this troubleshooting tree does not apply. The WHO Laboratory Biosafety Manual is for the people who write that list.

Inactivate the recovery broth you do not plate, along with the cuvettes, by the waste route already in force.

Hot rooms, lying displays and fogged cuvettes

A heat block set in a room that is already very warm can overshoot, especially if it was left on all day. Read the well. A block that is "close enough" at 48 Celsius is a killing step. In the other direction, a cheap block that never quite reaches the set point gives you a shock that never happened. Either way the plate is empty and the plasmid is innocent.

Cuvettes stored in a humid cold room fog when they meet warmer air. Wipe the outside dry, and let no film of water bridge the contacts. Condensation inside the gap is a bubble waiting to arc. Work quickly so the cells stay cold while you dry only the exterior. A power cut mid-session is a reason to discard an aliquot that warmed, not a reason to pulse it an hour later.

What to send with the question

Name the competence type, the strain, the marker, whether a known plasmid in low-salt water succeeded, whether an arc was reported, and what temperature the heat-block well actually held. Ask for the cell specification and for which cuvette the instrument programme was written. Put that on the quote request. A method can be discussed. Supplier protocol collections such as Promega protocols illustrate how those sheets are usually organised. They are not a voltage table to copy onto a different machine. The Addgene molecular biology reference is a glossary-level public companion for the words on those sheets.