Troubleshooting plates with no colonies

An empty plate is an investigation that stalls when four words are treated as one failure. Competent cell, ligation, selection, and transformation efficiency name different objects. You are deciding which object broke, using a control plasmid of known mass beside the new DNA. Colony screening has not started, because there is no colony to screen. The wider chain sits in plasmid cloning from insert to colony. This page defines the words inside the empty-plate case. It is research troubleshooting, not a kit insert.

What has to happen before a colony can exist

A colony here is the outgrowth of a cell that took up a plasmid, survived the antibiotic, and divided. The plasmid has to be a molecule that strain can copy, almost always a circle with an origin and a resistance gene. Linear fragments transform ordinary laboratory E. coli very poorly. Heat, salt, and the wrong drug interrupt the chain at different links. How those cells are grown and shocked is covered in bacterial culture and transformation. Use that note for the culture itself. Use this one when the agar stays bare.

The four terms, as this plate uses them

A competent cell is a bacterium treated so plasmid DNA can cross the envelope. Chemical competence is a cold, cation-based state. Electroporation is a pulse across a washed cell, and it demands very little salt in the DNA. Competence belongs to that aliquot and that afternoon. It is not a species name, and it does not survive being left warm. If you set the tube on a hot plate while you labelled agar, you no longer have the competent cells the box described.

Ligation is the enzymatic sealing of a nick between a 5-prime phosphate and a 3-prime hydroxyl where two ends can sit together. The usual enzyme class is T4 DNA ligase, and it needs ATP. No ligation means the circle was never finished. Incompatible overhangs, a blunt join that never found a partner, expired ATP in a buffer that was warmed all week, or a phosphatase placed on both the vector and the insert all present as no ligation. Gibson-style assembly is a different enzyme mix, but the glossary point is the same: if the join did not produce a circle, excellent cells still return an empty plate. Phosphatase belongs on the vector when the aim is to stop self-closure. On both partners, ligase has nothing to seal.

Selection is the match between the antibiotic in the agar and the resistance gene on the plasmid, at a concentration that stops untransformed cells of that host. The concentration belongs to that marker and that medium. A number copied from a different backbone is a guess. The wrong antibiotic is a perfect empty plate: the molecule may be flawless, and the marker cannot protect the cell from the drug you poured.

Transformation efficiency is colonies per microgram of a control plasmid of known mass, measured under stated conditions. It describes those cells and that control molecule. It does not describe your ligation, which is usually less abundant and often still in ligation buffer. A figure on a cell tube is the vendor's assay, not a prediction for today's join. Measure the control on the plates you are using.

Split the plate before you split the sequence

Include three tubes whenever you can: the new ligation or assembly, vector without insert, and a control plasmid resuspended as that transformation method requires. Spread each on agar that carries the antibiotic written on the backbone. Incubate for the time your strain note gives, then read the pattern. Do not lengthen the incubation and call a late haze a colony.

If the control plasmid is empty, stop debugging ends. Check the drug, the cells, and the heat. If the control grows and the new DNA does not, the cells can work and this molecule cannot: join, salt, or a toxic product. If vector-only is full and the ligation is empty, the backbone can recircularise and the insert reaction did not yield a recoverable circle. Follow the ligase or assembly sheet you opened. The Addgene restriction digest protocol is a public method note for the cut that came before.

Six ways the agar stays bare

Wrong antibiotic. The backbone says kanamycin and the plate says ampicillin, or the reverse. The control plasmid has to carry the plate's marker. Growth of a differently marked control on its own drug, beside silence on yours, is the answer. Pour the matching agar.

Cells left on a hot plate. A heat shock is a brief step at the temperature that cell protocol names. A tube parked on a heating block, or an aliquot left beside a hot plate while cuvettes are found, is a different treatment. Those cells die, and a control plasmid that shared the heat dies with them.

No ligation. The control plasmid is already a circle, so it forms colonies while your mix does not. Check compatible ends, ATP that has not been exhausted by warmth, and a phosphatase that was kept off the insert. Uncut vector would have given colonies. Linear DNA will not.

Toxic insert. Empty vector transforms and the insert construct does not, so competence and selection are working. A leaky promoter, a harmful protein, or a hostile backbone is the remaining explanation. Keep the empty-vector plate as the comparison and change host, copy number, or regulation.

Electroporation spark from salt. A flash, a pop, or a collapsed time constant means the sample arced. Ligation buffer is salty, the pulse dumps, and the cells die. A control plasmid in water electroporates on the same instrument. Desalt within the electroporation note you have, or switch that reaction to chemical competence.

Plates poured too hot. Cells spread onto agar that has not cooled are killed, and the plate stays empty. A sister plate that has cooled will still grow the control from the same tube. Adding antibiotic to molten agar that is still far too hot is a different error: the drug dies, and the symptom is growth everywhere. Emptiness after a hot spread means dead cells.

Safety

Recombinant plates are biological waste even when nothing grew. Dispose of agar as your committee requires. The WHO Laboratory biosafety manual, 4th edition is background, not an approval to transform a hazardous insert. A cloning-toxic insert is a fitness problem for the strain. Your institution decides containment.

Heat on the bench

In a hot room an ice bucket is water by the time the ligations are labelled. Cells that sat in that water have the same failure as a tube on a hot plate. Keep the aliquot frozen until the ice is ice. If you pour plates and spread them before the agar has cooled, you will record a burn as a cloning failure. Cool the plates, then transform.

What to ask for

Name the competent-cell class, chemical or electroporation, the control plasmid's marker, and the antibiotic on the backbone. Those items sit in the molecular biology catalogue. Say whether the control plate was empty too. Frame a later junction read with the Sanger sequencing enquiry reference. If a specified fragment would replace this ligation, the custom gene synthesis enquiry reference is only a specification prompt. Ask whether a quotation is possible on the quote request.