PCR not working? A troubleshooting checklist for no band

Updated October 30, 2026

An empty lane after PCR is one of the most common frustrations at the bench. The cause is usually simple, and checking things in the right order finds it fastest. Start with what the controls tell you, then work from the most likely causes to the least likely.

1. Read your controls first

• Is the ladder visible? If not, the problem is the gel, the stain or the imaging, not the PCR.
• Did the positive control work? A positive control (a template and primer pair known to work) separates "the reaction failed" from "this template or these primers failed".
  • Positive control also empty: suspect reagents, the master mix or the thermocycler program.
  • Positive control fine: suspect your template or your primers.
• No-template control should be empty. A band there means contamination, a different problem from this one.

2. Reagents and setup

• A missing component is the most common cause of all. Polymerase, primers or template left out of the mix. Writing a master mix table and ticking components off as you add them prevents it.
• Mg²⁺. Most polymerases need about 1.5–2.5 mM. Check whether your buffer already contains MgCl₂ or whether it must be added separately.
• Old or repeatedly thawed reagents. dNTPs and polymerase lose activity over many freeze–thaw cycles. Keep enzymes on ice or in a cooling block while working.
• Wrong volumes. Recalculate final concentrations. Primers at 0.2–0.5 µM and dNTPs at 0.2 mM each are typical.

3. Template

• Too little or too much. For a typical reaction, 1–10 ng of plasmid or 10–100 ng of genomic DNA is a good range. Too much template can inhibit the reaction as much as too little.
• Inhibitors. Carry-over of ethanol, phenol, guanidine salts, EDTA or heparin inhibits polymerases. A low A260/230 ratio is a warning sign. Try diluting the template 1:10: if the band appears, an inhibitor was the cause.
• Degraded DNA may still amplify short products but not long ones. Run the template on a gel to check its integrity.
• cDNA: confirm the reverse transcription worked and that the gene is expressed in your sample.

4. Primers

• Check the sequences against the template. Typos, a primer designed on the wrong strand or a reverse primer that was not reverse-complemented are surprisingly common.
• Annealing temperature too high. Recalculate the primer Tm and start about 3–5 °C below the lower Tm, or run a gradient PCR.
• Primer-dimers or hairpins can consume primers. A strong band below about 100 bp suggests dimers.
• Degraded or wrongly diluted stocks. Remake the working stock from the 100 µM stock.

5. Cycling conditions

• Extension time too short for the product. Taq needs about 1 minute per kb; many high-fidelity enzymes need 15–30 seconds per kb. Check your enzyme's datasheet.
• Too few cycles. 25–35 cycles is normal. Low-copy templates may need the upper end.
• Initial denaturation too short for hot-start enzymes, which need activation, often 2–3 minutes or longer.
• GC-rich targets (above about 65% GC) often fail. Adding 3–5% DMSO or 0.5–1.5 M betaine, raising the denaturation temperature, or using a polymerase made for GC-rich templates usually helps.
• Check the program actually ran, with the lid heated and the right block temperature. Program errors are easy to miss.

6. The gel

• Use an agarose percentage suited to your product size: very small products can run off a low-percentage gel, and large ones barely enter a high-percentage gel.
• Make sure the stain was added (or post-stain the gel), and load enough product: 5 µL of a 25 µL reaction is usually enough.
• Check the gel was run with the wells at the negative (black) electrode, so DNA moved into the gel rather than out of the wells.

A quick rescue plan

1. Repeat with a fresh master mix and a positive control.
2. Run the template at its current concentration and diluted 1:10.
3. Run a gradient of annealing temperatures across about 8 °C.
4. If the target is GC-rich, add DMSO or betaine.

Changing one variable at a time takes longer, but tells you what the actual problem was.