Technique hub

PCR protocols — versioned, with troubleshooting and fixes.

Q: Why are there no bands in my PCR?

If the positive control works but your reaction is blank, the annealing temperature is usually too high or the template is too dilute or degraded — lower the annealing 2–4 °C (or run a gradient) and re-quantify the template. If nothing amplifies at all, including the control, suspect a missing or inactive reagent: remake the master mix from fresh aliquots and check the Mg²⁺ concentration. GC-rich targets that won't amplify usually need DMSO or betaine plus a touchdown program.

Q: How do I get rid of nonspecific bands in PCR?

Extra bands almost always mean the annealing temperature is too low, so the primers are binding off-target — raise the annealing temperature or switch to a touchdown program where it starts high and steps down. A ladder-like smear instead points to too many cycles or too much template; drop to 25–30 cycles and use a hot-start polymerase. If the off-target binding persists, redesign the primers against a unique site.

Q: What causes primer dimers and how do I remove them?

Primer dimers are a bright band under ~100 bp formed when primers anneal to each other instead of the template, especially when the target is scarce. Redesign the primers to remove 3′ complementarity, lower the primer concentration, raise annealing stringency, and use a hot-start enzyme so extension can't start during setup. Adding more template also helps the real product out-compete the dimer.

Q: What are the steps of a PCR protocol?

Design and reconstitute primers, assemble a master mix (buffer, dNTPs, Mg²⁺, primers, polymerase), add template plus a no-template control, run an initial denaturation, then cycle denature → anneal → extend 25–35 times, finish with a final extension, hold/store, and confirm a single band on an agarose gel. Each step has a characteristic failure — annealing temperature governs specificity, and the NTC catches contamination.

Q: Do you have a PCR protocol PDF I can download?

Rather than a static PDF that's out of date as soon as someone changes the annealing temperature, jnar lets you import your own PCR protocol — drop in a methods PDF and turn it into a structured, versioned protocol with a visible fix history, so the cycling tweak that finally worked stays attached to the method. Your first AI conversion is free at /import.

PCR amplifies a specific DNA region through repeated cycles of denaturation, primer annealing, and extension. Most failures cluster in a few places: no bands from an annealing temperature that's too high or a stubborn GC-rich template, nonspecific bands from annealing that's too low, and primer dimers from poorly designed primers. The PCR protocols below keep those fixes visible — version by version.

Versioned & attributed protocols

Negative results recorded inline

Import your own with AI — first free

Running real-time PCR instead? See the qPCR protocols hub for Cq, amplification efficiency and standard-curve troubleshooting.

Standard v5

Step by step

A PCR, step by step — and where each one fails.

The classic endpoint-PCR workflow. Vendor pages list the steps; what they leave out is the failure that lives in each one. We keep both together.

Design & reconstitute primers

Pick primers with matched Tm (~58–62 °C), minimal self/cross-complementarity, and a unique target; resuspend to a known stock.

Most common failure Poor primer design is the root cause of both primer dimers and nonspecific bands.

Assemble the master mix

Combine buffer, dNTPs, MgCl₂, primers, and polymerase as a single master mix, then aliquot — set up on ice.

Most common failure Pipetting each reaction by hand drives well-to-well variability and contamination risk.

Add template DNA

Add a defined amount of clean template; include a no-template control (NTC) and a positive control.

Most common failure Too much template, or template carrying inhibitors, gives smearing or no product.

Initial denaturation

Hold at 94–98 °C to fully separate the strands before cycling.

Most common failure Under-denaturation of GC-rich template leaves it double-stranded → no amplification.

Cycle: denature · anneal · extend

Repeat denature → primer annealing → extension for 25–35 cycles.

Most common failure Annealing too low → nonspecific bands; too high → no bands at all.

Final extension

A single longer extension lets the polymerase finish partial products.

Most common failure Too short for long amplicons → truncated product and smearing.

Hold / store

Cool to 4–12 °C; move to the freezer if not run promptly.

Most common failure Leaving reactions warm too long degrades product and promotes nonspecific extension.

Run on an agarose gel

Size-separate against a ladder to confirm a single band at the expected size.

Most common failure Mistaking a <100 bp primer-dimer band for product, or using the wrong ladder.

Troubleshooting

PCR troubleshooting, grouped by what went wrong.

Five failure families cover almost every PCR that doesn't work the first time. Match the symptom, find the likely cause, apply the fix.

No bands / no product

Symptom	Likely cause	Fix
Blank lane, positive control works	Annealing temp too high; template too dilute or degraded	Lower annealing 2–4 °C or run a gradient; re-quantify and add more clean template
Nothing amplifies, control included	Missing or inactive reagent (polymerase, dNTPs, Mg²⁺)	Remake the master mix from fresh aliquots; verify Mg²⁺ concentration
GC-rich target won't amplify	Incomplete denaturation / strong secondary structure	Add 3–5 % DMSO or betaine; raise denaturation temp; use a touchdown program

Nonspecific / multiple bands

Symptom	Likely cause	Fix
Extra bands above/below the target	Annealing temp too low; primers bind off-target	Raise annealing or use touchdown; redesign primers for a unique site
Ladder-like smear of bands	Too many cycles / too much template	Drop to 25–30 cycles; reduce template; use a hot-start polymerase

Primer dimers

Symptom	Likely cause	Fix
Bright band <100 bp	Primers self/cross-anneal; primer excess	Redesign to remove 3′ complementarity; lower primer concentration; hot-start
Dimers compete out the real product	Low target abundance + dimer-prone primers	Increase annealing stringency; titre primers down; add more template

Smearing

Symptom	Likely cause	Fix
Vertical smear, no clean band	Excess template/cycles or degraded DNA	Reduce template and cycles; check DNA integrity on a gel before PCR

Contamination (band in the NTC)

Symptom	Likely cause	Fix
Product appears in the no-template control	Carry-over of amplicon or template into reagents	Use filter tips and a clean PCR area; aliquot reagents; remake the master mix

The moat

Every fix stays attached to the protocol.

The annealing temperature that finally gave a clean band, the DMSO that rescued a GC-rich template, the primer redesign that killed the dimer — on jnar that knowledge lives in the protocol, version by version. Not in someone's notebook, not in a folder of pcr_v3_FINAL.docx copies.

✓What changed and why, kept side by side
✓Negative results recorded as first-class data
✓Every change attributed via ORCID

How protocol version control works →

Endpoint PCR · GC-rich promoter v4

FIX HISTORY

Annealing 55 → 62 °C (touchdown)

Killed nonspecific bands · A. Moreau

Added 5% DMSO to the master mix

Fixed no-amp on GC-rich template · R. Tanaka

Primers redesigned, Tm matched at 60 °C

Removed primer dimers · K. Lindqvist

Initial published method

K. Lindqvist

− Anneal 55 °C, no additive

+ Touchdown 65→62 °C, 5% DMSO

In depth

Nonspecific bands & primer dimers, fixed properly.

Specificity in PCR is governed mostly by the annealing temperature. Set it too low and the primers tolerate mismatches, binding off-target and throwing extra bands; set it too high and they won't bind at all. The robust fix is a touchdown program: start a few degrees above the primer Tm and step down each cycle, so the earliest, most specific products dominate before lower-stringency cycles begin.

Primer dimers — that bright band under ~100 bp — form when primers anneal to each other rather than the template, and they're worst when the target is scarce. Redesign to remove 3′-end complementarity, drop the primer concentration, and use a hot-start polymerase so no extension happens during room-temperature setup. More clean template helps the real product win the competition.

A ladder-like smear is a different signal: usually too many cycles, too much template, or degraded DNA. Drop to 25–30 cycles, reduce input, and check DNA integrity on a gel first. And always read the no-template control — a band there means contamination, not a cycling problem, and no amount of temperature tuning will fix it.

On jnar, the touchdown program or additive that finally gave you a clean band is recorded against the protocol version, so the next person inherits it instead of rediscovering it.

Looking for a PCR protocol PDF?

A PDF goes stale the moment someone changes the annealing temperature. Import your methods PDF into jnar instead and get a structured, versioned PCR protocol with a living fix history — the cycling tweaks and their reasons stay together. Your first AI conversion is free.

Turn a PDF into a protocol →

Explore variants

Standard endpoint PCR PCR for GC-rich templates Colony PCR Long-range PCR Primer design & specificity PCR troubleshooting

PCR troubleshooting, answered.

Why are there no bands in my PCR?

If the positive control works but your reaction is blank, the annealing temperature is usually too high or the template is too dilute or degraded — lower the annealing 2–4 °C (or run a gradient) and re-quantify the template. If nothing amplifies at all, including the control, suspect a missing or inactive reagent: remake the master mix from fresh aliquots and check the Mg²⁺ concentration. GC-rich targets that won't amplify usually need DMSO or betaine plus a touchdown program.

How do I get rid of nonspecific bands in PCR?

Extra bands almost always mean the annealing temperature is too low, so the primers are binding off-target — raise the annealing temperature or switch to a touchdown program where it starts high and steps down. A ladder-like smear instead points to too many cycles or too much template; drop to 25–30 cycles and use a hot-start polymerase. If the off-target binding persists, redesign the primers against a unique site.

What causes primer dimers and how do I remove them?

Primer dimers are a bright band under ~100 bp formed when primers anneal to each other instead of the template, especially when the target is scarce. Redesign the primers to remove 3′ complementarity, lower the primer concentration, raise annealing stringency, and use a hot-start enzyme so extension can't start during setup. Adding more template also helps the real product out-compete the dimer.

What are the steps of a PCR protocol?

Design and reconstitute primers, assemble a master mix (buffer, dNTPs, Mg²⁺, primers, polymerase), add template plus a no-template control, run an initial denaturation, then cycle denature → anneal → extend 25–35 times, finish with a final extension, hold/store, and confirm a single band on an agarose gel. Each step has a characteristic failure — annealing temperature governs specificity, and the NTC catches contamination.

Do you have a PCR protocol PDF I can download?