Glycerol stocks of an expression clone

The expression that worked last month lives in a frozen vial, or it does not live anywhere. A glycerol stock is a clonal bacterial archive in a cryoprotectant, frozen so you can return to the same plasmid-bearing cells. It is not a purified protein, and it is not a sequence. This troubleshooting page is about how that archive becomes a mixture, a plasmid-free culture, or a gene you never meant to scale. It is research practice, not a strain-collection standard.

Freezer logic for biological samples is in storing biological samples from fridge to freezer. How a colony becomes a plasmid is in plasmid cloning from insert to colony. The protein gel you will run after the culture grows is in reading a protein gel.

A colony has to be one clone

Start from a single, well-isolated colony on a plate that contains the antibiotic matching the plasmid marker. A colony that touches its neighbour is two genomes until you restreak. Aseptic transfer matters more than the brand of cryovial. Flame or an equivalent sterile technique, a sterile tip or loop, and a tube you can label while your gloves are still clean are the equipment class. Work so that the lid is open briefly. A stock made from a swab of a whole plate averages every colony, including the ones that lost the insert and grew slowly.

Inoculate a small culture from that colony, grow it under selection, and only then mix with glycerol. Some laboratories pipette a colony straight into glycerol medium. That can work for a hardy strain and still hide a mixed pick. A short liquid culture lets you look at turbidity and gives you cells for a plasmid prep. Follow the route your lab already trusts. The decision is the same: one clone, under selection, then freeze.

Final glycerol is often discussed around 15 to 25 percent. Too little cryoprotectant and the freeze kills more cells than you expected. Too much glycerol is a different recipe from the one recovery conditions assume. Use your laboratory's method and record the final percentage, not a remembered splash. Sterile glycerol, mixed evenly, is the reagent class. Undissolved streaks of glycerol in the vial freeze as a gradient.

The master stays frozen

Freeze promptly at the temperature your archive uses, commonly an ultra-low freezer. A vial left overnight in a frost-free freezer that warms itself on a cycle is not on that method. Once frozen, the master is an archive. To start a culture, cool a needle or a sterile tip, scrape the surface, and return the master before the ice disappears. Thawing the master to pipette it "properly" puts the entire history of the clone through a freeze-thaw. Viability drops, and any contaminant you introduce is now in the only copy.

Make a working stock from the master on a planned day, and spend the working stock. When the working stock is low, revive once from the master and replace the working vial. Label master and working so a new colleague does not guess. Date both. A box of identical lids is how the wrong construct is expressed for a month.

Sequence before the large culture

Antibiotic in the agar is not a map. It selects for the marker. Cells can lose the insert and keep the resistance gene, rearrange a toxic coding sequence, or carry a mutation that appeared in the colony you picked. Before you commit a large expression, prepare plasmid from the archived clone and check the sequence across the insert and the junctions you care about. Compare it with the amino-acid sequence you think you are purifying. A perfect resistance phenotype with a frameshifted insert explains an empty gel better than any induction trick.

If sequencing disagrees with the map, stop. Do not scale the culture and hope the protein band appears. Return to a verified miniprep or remake the clone. Record the trace identifier beside the vial name so the freezer and the sequence file point at each other.

Expression from an unverified stock also confuses solubility work. An inclusion body of a truncated chain and an inclusion body of the full protein ask for different decisions. The gel cannot tell you the DNA was wrong if you never looked.

Contamination and satellites

Satellite colonies are the classic ampicillin failure. A beta-lactamase-secreting colony destroys antibiotic in a halo, and small colonies of cells that lack the plasmid grow in that halo. They look conveniently numerous. They are the wrong cells. Other antibiotics have different failure looks: a lawn when the dose is too low, or no colonies when the marker and the drug do not match. Read the plate. If satellites are present, pick only a large isolated colony of the expected morphology, or restreak onto a fresh plate poured recently enough that the antibiotic is still active. Ampicillin plates age. A plate that sat warm and damp for weeks is a different selective medium.

Contamination of the stock itself shows up later as a culture that smells wrong, a colony morphology that changed, or a plasmid prep that does not match the expected restriction or sequence pattern. Mixed morphology on a restreak from the vial means the archive is mixed. Do not pick "the one that looks right" from a mixed restreak and call the vial clean. Make a new stock from a verified single colony, or return to the DNA.

Turbid vials that were never cloudy, lids that were loose, and stocks made on a non-sterile bench are the usual routes in. Aseptic technique is the control. A preservative is not a substitute for it.

When the protein prep disagrees with the archive

A stock can be clonal and sequenced and still produce a pellet full of inclusion body. That is an expression outcome, not a glycerol failure. Troubleshoot induction, temperature and host. A stock that suddenly produces no band, after months of a reliable band, is when you go back to the sequence and the restreak. Do not retune the IPTG concentration on a culture that no longer carries the insert.

Quantification of the protein comes after you know which protein you grew. A Bradford number on a lysate from the wrong clone is a precise measurement of a mixture you did not intend.

Biosafety of an archive

Antibiotic-resistant strains are biological agents under your institution's rules. The biosafety manual frame from the World Health Organization does not replace local approval. Disinfect spills, do not pour cultures down a sink against policy, and label resistance markers on the box so the next person knows what they are thawing. This page is not a permit to store pathogens, and a research stock is not a product release.

Heat, power cuts, and a vial that arrived soft

Ultra-low freezers in warm, humid buildings frost up and fail when power fails. A stock that thawed and refroze is a spent master even if it still looks frozen in the morning. Quarantine it, revive a backup if you have one, and sequence whatever grows before you trust it. If you must ship a clone, prefer DNA or a properly packed frozen vial, and state that the receiver should not archive a vial that arrived thawed. Dry ice handoff belongs to the cold-chain practice you already use for samples. A glycerol stock left in a warm courier bag is a new, untrusted culture.

Write the construct, the antibiotic, the date, the sequence reference and the glycerol percentage on the vial and in the freezer index. Humidity peels paper labels. Ink on tape, plus a box map, survives.

What to include if someone else will grow the clone

Send the sequence, the marker, the final glycerol percentage you used, whether the master has been kept frozen, and the plasmid-prep check you already did. Say if expression from this exact stock produced soluble protein or inclusion bodies. Culture additives are reagent classes in the reagents and chemicals catalogue. Growing and purifying from a described clone can be discussed through the custom protein expression enquiry reference. Put the sequence status in the quote request so the work starts from a clone rather than from an antibiotic story.