TITLE: LAMP/RT-LAMP Reaction protocol
AUTHORS: Tamara Matute, Isaac Núñez, Fernan Federici

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to perform </span><span style = "font-weight:bold;">LAMP / RT-LAMP reactions with home-made reagents</span><span>, which means home-made buffers and positive controls, together with home-brewed BstLF and MMLV enzymes.</span></div><div class = "text-block"><span style = "font-weight:bold;">Home-brewed enzymes information:</span></div><div class = "text-block"><span style = "font-weight:bold;">BstLF </span><span>polymerase:</span></div><div class = "text-block"><span>expression and </span><a href="https://www.protocols.io/view/recombinant-protein-expression-and-purification-of-bksrkwd6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">purification protocol</span></a><span>. </span></div><div class = "text-block"><span>expression </span><a href="https://benchling.com/fernanfederici/f/lib_NJDXO9zf-alex_brown_plasmids/seq_lWgcPfoO-pet15b_bst-lf/edit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">plasmid sequence</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">M-MLV</span><span> reverse transcriptase:</span></div><div class = "text-block"><span>expression and </span><a href="https://www.protocols.io/view/recombinant-expression-and-purification-of-codon-o-bhcvj2w6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">purification protocol</span></a><span>. </span></div><div class = "text-block"><span>expression </span><a href="https://benchling.com/fernanfederici/f/lib_PdS1Nifz-public_seq_paper_dont_delete/seq_rNFedbX5-pet-19b_mmlv-rt/edit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">plasmid sequence</span></a><span>. </span></div><div class = "text-block"><span style = "font-weight:bold;">Positive control information</span></div><div class = "text-block"><a href="https://www.protocols.io/view/lamp-rt-lamp-covid-positive-control-bsknncve" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Sample preparation protocol here</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Testing</span></div><div class = "text-block"><span>We test this protocol in LAMP or RT-LAMP reactions with </span><a href="https://benchling.com/s/seq-75ss3ORzeIIdAdEoeXd8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">SARS-CoV-2 synthetic dsDNA or ssRNA</span></a><span> respectively. As shown in </span><span style = "font-weight:bold;">Figure 1</span><span>, the general performance of reactions with homemade reagents seems appropriate compared with the commercial standard option. No amplification was observed in non-template controls (NTC).</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/c3swhefe.jpg" /></div><div class = "text-block"><span>[1] </span><a href="http://paperpile.com/b/tgRxAv/XAC8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Zhang, Y. et al. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques69, (2020).</span></a></div></div>

[STEPS]
?. [Primers Stock]
Prepare 10X LAMP Primer Mix stock according to the following table:
?. [Primers Stock]
Prepare stocks (100μM) of each primer in nuclease-free water and store at –20°C for up to 2 years.
?. [Reaction]
Vortex and spin reaction mix.
on ice
If you need to add different samples; pipet   per reaction into desired reaction vessels and add   sample. Vortex and spin reaction mix. Always keep   to reduce spurious amplifications risk or sample degradation.
?. [Reaction]
Prepare reaction mix as described in the following table:.
[Always keep all components and reaction mix on ice.]
All the protocols for the preparation of the buffers are available here:
?. [Reaction]
Incubate at  for
65 °C
Time can be between 20 - 60 minutes depending on the specific assay. Specific temperature can vary according to the optimal temperature of the primer set used. The reaction can be carried out in any heater device from a water bath to real-time machines but the performance of the reactions can vary with respect to the temperature control.
?. [Reaction]
Incubate at  for .
65 °C
Time can be between 20 - 60 minutes depending on the specific assay. Specific temperature can vary accord to the optimal temperature of the primer set used. The reaction can be carried out in any heater device from a water bath to real-time machines but the performance of the reactions can vary a little accord the temperature precision control.
?. [Reaction]
Vortex and spin reaction mix.
on ice
If you need to add different samples; pipet   per reaction into desired reaction vessels and add   sample. Vortex and spin reaction mix. Always keep   to reduce spurious amplifications risk or sample degradation.