protocol with open access reagents

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Open access means freedom of information. With this protocol, anyone in the world can make their own RT-LAMP assays on a massive scale, producing most of the reagents themselves. Buffers for our open access assays are non-proprietary and their composition and preparation are listed here. Enzymes that power these reactions can be expressed and purified in any molecular biology lab in the world. A plasmid and four days of work is all it takes to produce enough enzymes for hundreds of thousands of RT-LAMP tests for SARS-CoV-2. The open access approach really enables large scale, decentralised and supply chain independent testing strategies. Universities, biohacker collectives, governmental research institutions and many more are encouraged to take matters into their own hands and establish their own, robust production pipelines all over the world.

Here's how it works...

Get plasmids

Order plasmids for enzyme production. Our plasmids are on Addgene for anyone to use.

Express and purify

Follow the published protocols to express enzymes for lots of reactions.

Benchmark and QC

Test your enzyme batches for activity and whole, assembled RT-LAMP reactions for sensitivity.

Test for covid-19

Help secure easy, fast, and cheap testing for your community.

Enzyme expression and purification

RT-LAMP reactions are catalysed by two enzymes, a reverse transcriptase that converts RNA to DNA, and a polymerase, that amplifies DNA in the presence of complementary primers. There are many enzymes that can do this, but non-patented wild-type enzymes best suited for this task are HIV-1 reverse transcriptase (HIV-RT) from the human immunodeficiency virus, and Bst polymerase Large Fragment, from the bacterium Bacillus stearothermophilus. Below, you can find the protocol to express these enzymes in E. coli and how to purify them. 

Buffers and solutions


Danube is an inactivation buffer based on the HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) protocol. Danube can be prepared as a 10X solution, enabling a larger sample input. Heat inactivation of samples by adding 1 volume of Danube to 9 volumes of sample and heating the mixture to 95 ºC for 5 minutes results in non-infectious samples with a complete inactivation of RNases.


  • Tris(2-carboxyethyl)phosphine hydrochloride solution, 0.5 M concentration, pH adjusted to 7.0 with ammonium hydroxide
  • Ethylenediaminetetraacetic (EDTA) solution, 0.5 M, pH 8.0
  • Betaine, 5 M solution
  • Proteinase K, 20 mg/ml, aqueous solution


  1. Calculate the volume of “Danube” inactivation solution you need based on the amount of samples you plan to inactivate.
    For performing RT-LAMP, we recommend inactivating at least 10 microlitres of sample, which requires 1 microlitre of Danube per sample. For performing bead-LAMP, we recommend inactivating at least 100 microlitres of sample, which requires 10 microlitres of Danube.
  2. Calculate the volume of needed reagents for number of samples you want to inactivate based on the following table
    ReagentStock concentration10x concentrationVolume to add for 1 ml
    TCEP0.5 M25 mM50 μl
    EDTA0.5 M10 mM20 μl
    Betaine5 M4.15 M830 μl
    Proteinase K20 mg/ml2 mg/ml100 μl
    Final Volume  1 ml
  3. Pipette the reagents together and mix by pipetting up and down or by vortexing and centrifuging.
  4. Use for inactivation of samples immediately.

Isothermal amplification buffer 10X

Isothermal amplification buffer is a well-known reagent in LAMP reactions. It provides optimal reaction conditions for a variety of polymerases derived from BstLF and the wild-type BstLF enzyme as well. A finished, ready to use 10X buffer can be purchased from New England Biolabs, but its composition is publicly known and the buffer itself is very easy to assemble. 


  • Tris hydrochloride, pH 8.8, 1.5 M solution
  • Potassium chloride, 3 M solution
  • Ammonium sulfate, 1 M solution
  • Magnesium sulfate, 1 M solution
  • Tween® 20
  • Nuclease-free water


  1. Prepare the materials either as pre-made stock solutions, or by making your own stock solutions.
  2. Calculate the volume of needed reagents for number of samples you want to inactivate based on the following table
  3. ReagentStock concentration10x concentrationVolume to add for 1 ml
    Tris hydrochloride1.5 M200 mM133 μl
    Potassium chloride3 M500 mM167 μl
    Ammonium sulfate1 M100 mM100 μl
    Magnesium sulfate1 M20 mM20 μl
    Tween® 20100 %1 %10 μl
    Nuclease-free water  570 μl
    Final Volume   
  4. Pipette the reagents together and mix by pipetting up and down thoroughly or by vortexing and centrifuging.
  5. Filter through a sterile 0.22 μm syringe filter and store at -20 ºC.

LAMP primer mix 10X

LAMP requires six primers for optimal functioning. That is six short DNA oligonucleotides to synthesise or order, the names are F3, B3, LF, LB, FIP and BIP. To make things easier, one can mix those in a pre-determined ratio to make a 10X primer mix to use with LAMP. You can make this mix with As1 primers against SARS-CoV-2 genome or against human ACTB transcript. Aliquot and label the finished primer mix with the name, date and name of the person who prepared it. We encourage to test every primer mix you receive for activity against the target template, especially when ordering from vendors you don’t have prior experience with. 







  1. Prepare your ordered oligonucleotides. If they are not resuspended, resuspend them in nuclease-free water for 100 μM stock concentration.
  2. In a clean and template-free workspace, such as in a PCR hood, mix the oligonucleotides in the order outlined by the table below. Make sure to add the components from largest volume to the smallest volume.
    Oligo nameAdd for 100 μl of mix
    F32 μl
    B32 μl
    LF4 μl
    LB4 μl
    FIP16 μl
    BIP16 μl
    nuclease-free water56 μl
  3. Mix the finished primer mix by thoroughly pipetting up and down or by vortexing and briefly spinning down in a microfuge.
  4. Distribute the finished primer mix into Eppendorf tubes into conveniently sized aliquots, label the tubes and store at -20 ºC.

RT-LAMP protocol

When you have the enzymes, primer mix and the sample inactivation reagent, it’s time to assemble the reactions themselves. Make sure to test your reactions on a known positive sample dilution first to assess the specificity and sensitivity of your assays. Expressing and purifying your own enzymes is not trivial, but the payoff is tremendous. If you are expressing your enzymes and having issues with the resulting reactions, please contact us for help in troubleshooting. Please consider the following before you start: 


  1. Prepare “Danube” 10X inactivation solution according to the protocol above.
  2. In a BSL-2 workspace with extra precautions taken against respiratory viruses, mix 10μl of Danube inactivation solution with 90 μl of sample.
    [CRITICAL] Make sure to adhere to the safety precautions for handling potentially infectious SARS-CoV-2 samples outlined by applicable health authorities in your jurisdiction.
  3. Heat-inactivate the mixture of sample and inactivation solution at 95°C for 5 minutes.
  4. Cool the inactivated samples on ice or at room temperature for immediate use or store at -20°C.
    [PAUSE] Samples can be stored at -20°C at this point for up to four weeks, potentially longer. Inactivated samples can now be handled in BSL-1 settings.

RT-LAMP reaction assembly (BSL1 WORKSPACE)

  1. Calculate your reagent consumption for assembling the RT-LAMP reaction master mix according to the table below. Multiply the last column by the number of samples you plan to test + 2 for a positive and negative control. Multiply this number by 1.1 to make sure you prepare an excess to compensate for pipetting error.
    Reagent Stock concentration Final concentration To add per reaction
    Isothermal amplification buffer 10X 1X 1 μl
    dNTP mix 25 mM 1.4 mM 0.56 μl
    dUTP 100 mM 0.7 mM 0.07 μl
    Magnesium sulfate 100 mM 6 mM 0.6 μl
    As1 primer mix 10X 1X 1 μl
    HNB dye, trisodium salt 20 mM 0.12 mM 0.06 μl
    Syto9 fluorescent dye (optional) 100 μM 2 μM 0.2 μl
    Betaine 5 M 0.4 M 0.8 μl
    HIV-RT enzyme 0.55 mg/ml 0.00275 mg/ml 0.05 μl
    BstLF enzyme 0.6 mg/ml 0.02 mg/ml 0. 33 μl
    Thermolabile UDG enzyme 1 U/μl 0.02 U/μl 0.2
    Nuclease-free water     to a total of 8 μl
    Final Volume     8 μl
  2. Prepare the RT-LAMP reaction master mix by mixing the reagents in an Eppendorf tube on ice. Add the enzymes last.
    [CRITICAL] Make sure to add the enzyme components last! They will otherwise denature and you will experience decreased assay performance or no activity at all.
    [CRITICAL] Assemble the RT-LAMP reaction master mix on ice.
  3. Mix the reagents by thoroughly pipetting up and down or by vortexing and spin down in a microfuge.
  4. Dispense the reaction mix into PCR strips or into a 96-well plate on ice. Each well should receive 8 μl of the reaction mix.
  5. On ice, add 2 μl of heat-inactivated sample treated with Danube buffer to RT-LAMP reaction mix and mix the reaction by pipetting up and down 10 times. Repeat for all samples that are to be tested, and include a proper positive and negative control. Seal PCR strips by capping them, and seal 96-well plates by applying a plastic transparent plate seal.
    [CRITICAL] This step should be done in a separate work area where no reagents are handled.
    [CRITICAL] If performing colorimetric detection, take an image of the PCR strips or 96-well plate with the mastermix and samples.
  6. Transfer the reactions to a suitable stable heat-source such as a heat block, thermocycler or a water bath. Incubate the reactions at 63°C for 35 minutes.
    [CRITICAL] For highest sensitivity and specificity, make sure you transfer the sample from ice to a preheated, 63°C environment.
    [OPTIONAL] For real-time fluorescence data acquisition in a qPCR thermocycler, we typically perform 35 cycles at 63°C with 1-minute cycle length and reading at the end of each cycle. Syto9 fluorescent dye requires data acquisition using a standard FAM or SYBR filter (494nm/518nm absorption/emission).
  7. After 35 minutes run-time, remove reactions and allow to cool briefly at room temperature. Then proceed to inspect reactions visually. Negative reactions are purple while positive reactions are sky-blue.
  8. Discard the reactions immediately after noting down the results.
    [CRITICAL] If performing colorimetric detection, take a second image of the PCR strips or 96-well plate under the same conditions as you took the first one after the reaction.

Readout methods

RT-LAMP color readout

Visual colorimetric

Hydroxynaphtol blue (HNB) is used in RT-LAMP reactions that do not include any purification step. The dye changes colour from purple to sky blue upon presence of target sequence and amplification. This can be visualised with the naked eye, and reactions can be scored directly after taking them out of the heating device.

RT-LAMP app enhanced color readout

App-assisted colorimetric

For people who find the colour change of HNB difficult to see with the naked eye, we recommend the use of a simple web app. Developed by Andrew Straw to increase the colour difference between positives and negatives and make the readout much simpler. Try it out on colorimetry.net

RT-LAMP fluorimetric detection


Amplification of DNA in RT-LAMP reactions can also be read out through the use of fluorescent intercalating dyes. These dyes, such as Syto9 or the NEB LAMP dye, enable the user to either monitor the progress of the reaction in real time through the use of a qPCR machine, or end-point detection with a plate reader or a similar fluorescence detection device.

Premixing information

Assembling each master mix for every testing session from scratch is not necessary, however freezing prepared master mixes increases the risk of running into sensitivity or specificity issues. We’ve tested the possible combinations and now we can tell you what you can and can’t premix for easier reaction assembly without compromising on the quality of the tests. 

LAMP reaction buffer premix

ReagentStock concentration To add per 100 reactions (10µl size)
Isothermal amplification buffer10X 100 μl
dNTP mix25 mM 56 μl
dUTP100 mM 7 μl
Magnesium sulfate100 mM 60 μl
As1 primer mix10X 100 μl
HNB dye20 mM 6 μl
Syto9 fluorescent dye (optional)100 μM 20 μl
Betaine5 M 80 μl
nuclease-free water  321 μl
total  750 μl

Add 7.5 µl of this mix per 1 reaction.

Store at -20 °C.

LAMP enzyme premix

ReagentStock concentrationAmount per reactionTo add per 100 reactions (10µl size)
HIV-RT enzymevariable75 ngvariable
BstLF enzymevariable200 ngvariable
Thermolabile UDG enzyme (NEB)1 U/μl0.2 U20 μl
storage buffer  to a total of 50 µl 
total   50 µl

Add 0.5 µl of this mix per 1 reaction.

The volumes for enzymes are listed as “variable” since each enzyme purification yields a different concentration of enzyme. We recommend measuring the yield you get from your purifications and make the calculations yourself, so as to reach 75 ng of reverse transcriptase and 200 ng of DNA polymerase, respectively. Then multiply the volumes for 75 ng and 200 ng of enzyme, respectively, by 100 to fill in the table. Add storage, as described in the enzyme expression and purification protocol on the open access protocol pages, to a total of 50 µl. 

Store at -20 °C.

The premixes for open-access RT-LAMP enable you to have two tubes in your -20 °C freezer and to assemble reactions easily using 7.5 µl of LAMP reaction buffer premix and 0.5 µl of LAMP enzyme premix per 10 µl reaction. We’ve observed their stability to be unchanged after 1 week’s storage at -20 °C, but they can most likely last for longer. It is crucial to work quickly with the enzyme mix and to work with it on ice or in the cold. 

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