Standard PCR Setup and Thermocycling
Foundational Polymerase Chain Reaction procedure for amplifying specific DNA sequences from a template. Covers master-mix assembly, thermocycling parameters, and essential controls (no-template, positive control, replicates). Atomic technique for procedure 56 (COI Fish Barcoding) and any downstream procedure requiring DNA amplification. Written for a novice audience; assumes familiarity with pipetting but not with PCR.
Version History
Version 0.1.1 Latest
Effective: 2026-04-20Cross-reference fixes: agarose-gel refs 68→73, Chelex ref 68→71, SPRI ref 70→74, Sanger ref 71→75. All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0 Viewing
Effective: 2026-04-20Initial release. Foundational amplification technique. PCR patents expired in the early 2000s; procedural facts are field-standard and uncopyrightable. Fresh original prose.
Procedure Details
- Not a hazardous procedure inherently — no toxic reagents in standard PCR.
- Pipetting hygiene. Filter tips throughout; dedicated pre-PCR area; glove changes when moving between sample types to prevent cross-contamination.
- No-template controls (NTC) are mandatory. The #1 failure mode of PCR is cross-contamination; the NTC catches it.
- Post-PCR amplicon is a contamination risk. Never open PCR tubes in the pre-PCR area — amplicons aerosolise and seed future NTC positives.
- UV use (if gel imaging used after PCR). See procedure 68 (Agarose Gel Electrophoresis) for UV-related safety.
Reagent preparation:
- Master mix. Use a 2X commercial PCR master mix (NEB OneTaq, NEB Q5, Taq polymerase homebrew from LibreBiotech procedure 59, or equivalent). 2X means the enzyme, dNTPs, buffer, and MgCl₂ are already mixed at 2× working concentration — just add primers, template, and water to reach 1× final.
- Primers. Order from IDT or equivalent. Standard 25-nmol scale at 100 µM stock, dilute working stocks to 10 µM for routine use. Store primer stocks at −20°C in single-use aliquots.
- Template. From DNA extraction (e.g. procedure 68 Chelex) at 5–50 ng/µL. Do NOT use undiluted if inhibited.
- Nuclease-free water. Commercial (Thermo, Ambion) or double-autoclaved Milli-Q.
- Sterile PCR tubes. 0.2 mL individual tubes or 8-tube strips. Never reuse.
Bench prep:
- Pre-PCR area: dedicated pipettes, fresh filter tips, gloves, no amplicon residue. Bleach-wipe surfaces before and after.
- Thermocycler programmed with the specific cycling profile before you start pipetting.
- Ice bucket for all reagents (especially master mix and primers).
Mental model: PCR amplifies a specific DNA segment by repeating a three-step cycle: (1) denature dsDNA to ssDNA at ~95°C, (2) anneal primers to their complementary sequences at a temperature specific to the primer pair (typically 50–65°C), (3) extend the primers with a polymerase at ~72°C. Each cycle doubles the target copy number; 30 cycles gives ~10⁹-fold amplification.
Designing a PCR:
- Volume: 25 µL standard; 10 µL for cost savings; 50 µL for downstream sequencing.
- Cycles: 30 standard; 25 for abundant template (avoids non-specific bias); 35 for scarce template (low-copy DNA, low-quality extract).
- Annealing temp: primer-specific. Use the primer vendor's Tm calculation or rough rule (Tm ≈ 2×(A+T) + 4×(G+C)), then set annealing = Tm – 5°C.
- Extension time: depends on amplicon size and polymerase. Taq: ~60 s per kb; Q5: ~30 s per kb; Vent: ~60 s per kb.
- Setup (15 min): thaw reagents on ice, mix master mix, aliquot, add samples.
- Thermocycling (~1.5–2.5 h): depending on cycling profile. Mostly hands-off.
- Post-PCR storage: 4°C short-term (hours), −20°C longer-term (weeks).
-
Microcentrifuge
Centrifugation
Specs: ≥13,000 × g
For brief spin-downs -
Micropipette
Liquid handling
Specs: P2, P20, P200; filter tips mandatory
Dedicated pre-PCR set -
Vortex mixer
Mixing
Specs: Standard benchtop
For reagent mixing — never for PCR tubes after template added -
Thermal cycler
Thermal regulation
Specs: Programmable ramp, heated lid, 0.2 mL tube format
Pi-controlled OpenPCR-compatible or commercial
-
Primer (custom)
Primer
Qty: 0.5 µL of 10 µM stock
Design-specific; final 0.2 µM in reaction -
Primer (custom)
Primer
Qty: 0.5 µL of 10 µM stock
Design-specific; final 0.2 µM -
Nuclease-free water
Reagent
Qty: 10.5 µL per rxn
To reach 25 µL final -
PCR master mix
Reagent
Qty: 12.5 µL per 25 µL rxn
NEB OneTaq 2X, NEB Q5 2X, or LibreBiotech procedure 59-derived cellular reagent Taq
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
reaction_volume_ul |
number | — |
25
|
microliter (UO:0000101) | PCR reaction volume. 25 µL standard; 10 µL for cost savings; 50 µL for sequencing downstream. |
cycle_count |
number | — |
30
|
— | Number of cycles. 30 standard; 25 for abundant template; 35 for scarce template. >40 risks non-specific products. |
annealing_temp_c |
number | — |
55
|
degree Celsius (UO:0000027) | Annealing temperature. Primer-pair specific. Rule of thumb: Tm - 5°C where Tm is the primer melting temperature. |
annealing_time_s |
number | — |
30
|
second (UO:0000010) | Annealing duration. 30 s standard; shorter (15 s) for Q5-type polymerases. |
extension_time_s_per_kb |
number | — |
60
|
second (UO:0000010) | Extension time per kb of amplicon. 60 s/kb for Taq; 30 s/kb for Q5; 60 s/kb for Vent. |
template_volume_ul |
number | — |
1
|
microliter (UO:0000101) | Template DNA volume. 1 µL (of 5–50 ng/µL extract) standard. More for scarce template; less for abundant. |
Procedure Steps (Version 0.1.0)
Program the thermocycler with the cycling profile: 95°C × 2 min (initial denaturation) → [95°C × 30 s denaturation, Tm-5°C × 30 s annealing, 72°C × (60 s/kb amplicon) extension] × 30 cycles → 72°C × 5 min (final extension) → 4°C hold.
Thaw master mix, primers, template, and nuclease-free water on ice. Do NOT thaw at room temperature.
Vortex each reagent briefly (2–3 s) and spin down in a microcentrifuge. Brief, not vigorous — enzymes are fragile.
In a clean 1.5 mL tube, prepare a master mix for (n+1) reactions where n = number of samples + 1 no-template control + 1 positive control. The +1 accounts for pipetting loss. Example for 5 reactions: 30 µL (5×6×1 reaction volume factor) working volume.
Per 25 µL reaction, mix: 12.5 µL 2X PCR master mix + 0.5 µL forward primer (10 µM) + 0.5 µL reverse primer (10 µM) + 10.5 µL nuclease-free water. Add these components in this order to a pooled master mix — DO NOT add template yet.
Vortex the pooled master mix briefly; spin down.
Aliquot 24 µL of pooled master mix into each labelled 0.2 mL PCR tube or strip well. Keep strips on ice while adding.
Add template: 1 µL of sample DNA to sample tubes. Add 1 µL of nuclease-free water to the NTC tube. Add 1 µL of known positive-control template to the positive-control tube. Use a fresh tip for every tube.
Cap tubes firmly. Briefly spin the PCR strips in a microcentrifuge (5–10 s at low speed) to settle reagents to the bottom. Do NOT vortex PCR reactions — mixing is adequate from pipetting.
Transfer tubes to the pre-heated thermocycler. Close lid firmly. Start the cycling program.
Monitor the thermocycler briefly to confirm cycling has started; then wait for completion (~1.5–2.5 hours depending on profile).
At cycle completion, tubes are at 4°C hold. Retrieve promptly — extended 4°C exposure does no harm but the faster you process, the better.
Record the PCR run in LibreBiotech: Process record with procedure_version, thermocycler log (export from the Pi controller if using open hardware), primer identifiers, template source, and NTC result. Attach these to the Process as annotations. The PCR products become Samples downstream, each linking back to this Process.
Verify products by agarose gel electrophoresis (procedure 68) before downstream use.
Store PCR products at 4°C overnight; −20°C for longer-term.
Expected outcome. Amplicon (PCR product) of the expected size. Verify by agarose gel electrophoresis (procedure 68). Expected band: single sharp band at the predicted amplicon size; no band in NTC; positive control gives expected band.
Storage of PCR products. 4°C for 1–2 weeks; −20°C for months. Do not freeze-thaw repeatedly (< 5 cycles).
Downstream uses. Direct gel load (procedure 68); SPRI cleanup (procedure 70) before Sanger submission (procedure 71); direct sequencing of clean single-band products.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No band on any sample | PCR failure — reagents, thermocycler, or sample | Test a known-good positive control; verify thermocycler temperature; check master mix not expired |
| Band in NTC | Cross-contamination | Remake all reagents in fresh tubes; clean bench; check thermocycler block for residue |
| Multiple bands (non-specific) | Annealing temp too low | Raise annealing temp 2–3°C; or add DMSO/betaine for difficult templates |
| Smear instead of band | Too much template; too many cycles | Dilute template 1:10; reduce cycles to 25 |
| Very faint band | Insufficient template; low enzyme activity | Larger template volume; fresh enzyme aliquot |
| Different band than expected size | Wrong primers; mispriming | Verify primer sequences; run mock on control template |
References
- Mullis KB, Faloona FA (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–50. (Original PCR method paper). DOI paper
- Saiki RK et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–91. (Taq + thermostable PCR). DOI paper
- Sambrook J, Russell DW (2001). Molecular Cloning: A Laboratory Manual, 3rd edition. Cold Spring Harbor Laboratory Press. (Standard reference for PCR practice). book