Read the gel first. No band, a smear, extra bands and a fuzzy band under 100 bp each point to a different change, and the positive control tells you whether to trust the mix.
Range: Molecular biology reagents3 minute readUpdated
| What you see | Likely cause | Change first |
|---|---|---|
| No band, control also fails | Mix component or cycling | Remake the mix; check the program |
| No band, control works | Template, primers or annealing | Lower annealing by 3–5 °C; check template |
| Smear | Too much template or too many cycles | Dilute template; reduce cycles |
| Several sharp bands | Non-specific priming | Raise annealing; gradient PCR |
| Fuzzy band under 100 bp | Primer dimers | Hot-start, less primer, redesign |
| Band in the no-template control | Contamination | New water and primers; clean workspace |
Primer dimers form when primers anneal to each other, especially when their 3′ ends are complementary. They run as a diffuse band below about 100 bp. Lower the primer concentration, usually to 0.1–0.5 µM each, set up on ice or use a hot-start enzyme, and redesign primers if dimers persist. In a qPCR run, the same problem appears as an extra melt peak; see the qPCR troubleshooting guide.
A band in the no-template control means amplified product or template got into a reagent. Discard the water and primer working stocks, use filter tips, and keep setup physically separate from where tubes are opened after PCR. If you plan to clone the product, see the cloning workflow. Polymerases and ladders are in the molecular biology range.
If the positive control also fails, remake the mix and check the cycling program. If it works, try a lower annealing temperature, a longer extension and a diluted template in case of inhibitors.
Too much template, too many cycles, degraded template or excess magnesium or enzyme. Diluting the template and reducing the cycle number are the first changes to make.
Use a hot-start polymerase, lower the primer concentration, raise the annealing temperature and redesign primers whose 3′ ends are complementary.
Start about 3–5 °C below the lower primer Tm and refine it with a gradient. Some high-fidelity polymerases use their own recommended calculation, so check the datasheet.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Cloning systems, DNA molecular weight standards and DNA polymerases for cloning, PCR and gel analysis.
Molecular biology
A DNA ladder is only useful if its bands bracket your fragment on the gel you are running. This guide matches ladder ranges to agarose percentages and covers loading, staining and quantification.
4 minute read
Molecular biology
Cloning is a chain of small steps, and most failures trace back to one of them. Here is the whole chain, the reagent each step needs, and how to check you have the right clone before you build on it.
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qPCR / RT-PCR
End-point PCR tells you whether a sequence is there, qPCR tells you how much, and RT-qPCR does the same for RNA. Here is what changes between them and the reagent list for each.
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