LAMP Reaction Setup and Thermal Incubation

Loop-mediated isothermal amplification (LAMP) workflow — uses four to six primers and a strand-displacing Bst polymerase to amplify target DNA at a single temperature (60–65°C) in 30–60 minutes, without a thermocycler. Ideal for field-deployable diagnostics, DIY-bio settings, and eDNA / pathogen-detection work. Uses LibreBiotech procedure 66 (Bst-LF cellular reagent) as the polymerase source. Written for a novice audience; pairs naturally with procedure 79 (pH-indicator visual readout) for equipment-free detection.

measurement

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Version History

Version 0.1.1 Viewing Latest

Effective: 2026-04-20

Cross-reference fix: LAMP pH-readout ref 79→78. 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

Effective: 2026-04-20

Initial release. LAMP technique (Notomi et al. 2000) with LibreBiotech-specific integration: uses procedure 66 Bst-LF cellular reagent. Fresh original prose from standard LAMP practice; foundational references cited.

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Procedure Details

Safety & Hazards

Contamination risk is the #1 LAMP failure mode. LAMP amplifies billions of copies per reaction; the amplicon aerosolises and seeds false positives in subsequent reactions. Strict pre-LAMP / post-LAMP workflow separation is mandatory: dedicated pipettes, dedicated pipette tips, dedicated benches, gloves changed between reagent setup and amplicon handling. Open post-LAMP tubes in a different room from pre-LAMP reagent preparation where possible.
Isothermal heating. 60–65°C heat block. Standard burn hazards.
Pipetting hygiene. Filter tips throughout; no-template controls (NTC) mandatory on every run.
Not a clinical assay. Research and educational use only. Do not use LAMP output for medical decisions without proper clinical validation.

Preparation Notes

Reagents:

Bst-LF polymerase — LibreBiotech procedure 66 cellular reagent (rehydrated at reaction time), or commercial alternative (NEB Bst 2.0, NEB Bst 3.0, LavaLAMP).
Six primers per assay:
- F3 (forward outer), B3 (backward outer) — 0.2 µM final each
- FIP (forward inner), BIP (backward inner) — 1.6 µM final each
- LF, LB (loop primers, optional — accelerate amplification ~2-fold) — 0.8 µM final each
dNTPs — 1.4 mM each final
MgSO₄ — 6–8 mM final
Reaction buffer — isothermal amplification buffer (20 mM Tris-HCl pH 8.8 + 10 mM (NH₄)₂SO₄ + 50 mM KCl + 0.1% Tween-20); commercial buffers work identically
Betaine (optional, recommended for GC-rich targets) — 0.4–1.0 M final
Nuclease-free water — top up to 25 µL final

Equipment:

60–65°C heat block or water bath. The specific temperature depends on the primer set's melting profile; 65°C is the default.
Sterile 0.2 mL PCR tubes.
Dedicated pre-LAMP pipettes.

Mental model. LAMP uses two "outer" primers (F3, B3) and two "inner" primers (FIP, BIP) to generate a dumbbell-shaped starting structure that self-primes exponential strand-displacing amplification. Loop primers (LF, LB) optional — they target the loop regions of the dumbbell and accelerate the reaction. The Bst-LF polymerase displaces strands rather than digesting them, so no denaturation step is needed — everything happens at one temperature.

Timing

Setup (15 min): rehydrate Bst-LF, thaw primer/dNTP stocks, mix master mix, aliquot reactions.
Incubation (30–60 min): single-temperature 60–65°C. Hands-off.
Readout (5 min): visual pH change (procedure 78), fluorescence, turbidity, or gel (procedure 73).
Total: ~45–75 min per run.

Equipment (Catalog) 4

Microcentrifuge Centrifugation
Specs: ≥13,000 × g for brief spins
Consolidate tube contents
Micropipette Liquid handling
Specs: P2, P20, P200; filter tips; dedicated pre-LAMP set
Dedicated pipettes are the single most important contamination-control measure
Vortex mixer Mixing
Specs: Standard benchtop
For reagent mixing — NEVER for LAMP tubes after template added
Heat block Thermal regulation
Specs: 60–65°C capable, ±1°C
Dry heat block preferred; water bath acceptable

Materials (Catalog) 9

Primer (custom) Primer
Qty: 0.5 µL of 10 µM per 25 µL rxn
Design-specific; 0.2 µM final
Primer (custom) Primer
Qty: 0.5 µL of 10 µM per 25 µL rxn
Design-specific; 0.2 µM final
Primer (custom) Primer
Qty: 0.4 µL of 100 µM per 25 µL rxn
Design-specific; 1.6 µM final
Primer (custom) Primer
Qty: 0.4 µL of 100 µM per 25 µL rxn
Design-specific; 1.6 µM final
Primer (custom) Primer
Qty: 0.2 µL of 100 µM (optional)
Accelerates amplification ~2× when present
Primer (custom) Primer
Qty: 0.2 µL of 100 µM (optional)
Pair with LF primer
Buffer Reagent
Qty: 2.5 µL 10× per 25 µL rxn
20 mM Tris-HCl pH 8.8 + 10 mM (NH4)2SO4 + 50 mM KCl + 0.1% Tween-20
Nuclease-free water Reagent
Qty: 7.5 µL per rxn
Top up to 25 µL
PCR master mix Reagent
Qty: varies
Bst-LF cellular reagent from LibreBiotech procedure 66, or commercial NEB Bst 2.0/3.0

Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns

Name	Type	Required	Default	Unit	Description
`reaction_temp_c`	number	—	`65`	degree Celsius (UO:0000027)	Isothermal reaction temperature. 65°C default for Bst-LF; range 60–65°C; primer-design-specific.
`reaction_time_min`	number	—	`45`	minute (UO:0000031)	Incubation duration. 30 min for abundant template; 45 min standard; 60 min for low-copy detection.
`reaction_volume_ul`	number	—	`25`	microliter (UO:0000101)	Reaction volume. 25 µL standard; scales down to 10 µL or up to 50 µL without altering chemistry.
`mgso4_conc_mM`	number	—	`6`	—	MgSO₄ final concentration. 6 mM default; optimise 4–10 mM for difficult templates.
`betaine_conc_M`	number	—	`1`	—	Betaine final concentration. 1 M standard for GC-rich targets; 0 for AT-rich (omits betaine from master mix).
`loop_primers_used`	boolean	—	`true`	—	Whether LF/LB loop primers are included. True ~2× acceleration; False (core 4-primer set only) simpler design.

Procedure Steps (Version 0.1.1)

Program or pre-heat the heat block to 65°C (default for Bst-LF); adjust to 60°C or 63°C if primer design dictates.

Prepare a primer mix: combine F3, B3, FIP, BIP, and optionally LF, LB at 10× their final reaction concentrations (2 µM F3/B3, 16 µM FIP/BIP, 8 µM LF/LB). Store aliquots at −20°C; thaw on ice before use.

Thaw dNTPs, MgSO₄, betaine (if used), reaction buffer, and water on ice.

Rehydrate a Bst-LF cellular-reagent aliquot (procedure 66 output) by adding 25 µL of the master mix directly to the aliquot tube — the dried bacterial cells release active enzyme during the first minute of incubation.

In a clean 1.5 mL tube, prepare a master mix for (n+1) reactions (n = samples + NTC + positive control). Per 25 µL reaction: 2.5 µL 10× primer mix, 3.5 µL dNTPs (10 mM each stock for 1.4 mM final), 3 µL MgSO₄ (50 mM stock for 6 mM final), 5 µL betaine (5 M stock for 1 M final, optional), 2.5 µL 10× reaction buffer, 7.5 µL water. DO NOT add Bst-LF or template yet.

Vortex the master mix briefly; spin down.

Aliquot 23 µL of master mix to each LAMP tube (which contains the rehydrated Bst-LF cellular reagent).

Add 2 µL of template to each tube. Sample tubes: 2 µL sample DNA. NTC tube: 2 µL water. Positive control: 2 µL known template. Use fresh tips for every tube.

Cap tubes firmly. Briefly spin down in a microcentrifuge (5 s at low speed) to consolidate contents.

Transfer tubes to the pre-heated 65°C heat block. Close the lid.

Incubate for 30–60 minutes. Shorter (30 min) for strong-positive samples; longer (60 min) for detection of low-copy targets.

Remove tubes. Immediately transfer to a post-LAMP handling zone — opening tubes at the bench where setup occurred will contaminate future runs.

Visualise amplification by the chosen readout method: pH indicator (procedure 78), fluorescence, turbidity, or agarose gel (procedure 73).

Record the LAMP run in LibreBiotech: Process record with procedure_version, template source, primer set, incubation parameters, readout method, and NTC result. Attach readout images/data. Every Sample becomes linked to this Process for downstream data integrity.

Completion Notes

Expected outcome. Successful LAMP: amplicon detectable in positive-control tubes (ladder-like bands on gel, fluorescence rise, pH change, turbidity). NTC: no amplification by any readout method. Sample tubes: amplification if target present, none if absent.

Readout choice:

pH indicator (procedure 78): fastest, cheapest, equipment-free. Recommended for field use.
Fluorescence (SYBR Green I, EvaGreen): sensitive, quantitative with a real-time instrument.
Turbidity (LAMP by-product Mg₂P₂O₇ precipitate): visible cloudiness; legacy readout.
Agarose gel (procedure 73): gold standard for method development; shows characteristic LAMP ladder pattern rather than single band.

Troubleshooting.

Symptom	Likely cause	Fix
NTC shows amplification	Cross-contamination (very common)	Dedicated pre-LAMP / post-LAMP zones; fresh pipettes; UV-sterilise benches; remake reagents
No amplification even with positive control	Bst-LF inactive; primers degraded; wrong temperature	Verify Bst-LF activity (procedure 66 QC); fresh primer aliquots; verify heat block temperature
Weak amplification (takes >60 min)	Missing loop primers; sub-optimal Mg²⁺	Add LF/LB loop primers; optimise MgSO₄ to 6–8 mM range
Amplification in sample but NTC-adjacent tubes are contaminated	Aerosol during tube opening	Seal tubes before leaving heat block; open away from setup area
Primer-design issues (no amplification on known-positive)	Primer design / target mismatch	Redesign primers with PrimerExplorer V5 (Eiken); verify template sequence

References

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63. (Foundational LAMP paper). DOI paper
Tomita N, Mori Y, Kanda H, Notomi T (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3(5):877–82. DOI paper
LibreBiotech procedure 66 — Bst-LF Cellular Reagent Production (polymerase source). Link protocol