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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the preparation of </span><span style = "font-weight:bold;">reaction buffer</span><span> for homemade LAMP / RT-LAMP reactions which involves the use of home-brewed BstLF and MMLV enzymes. It also contains the instructions to prepare the enzyme </span><span style = "font-weight:bold;">storage buffers</span><span style = "font-weight:bold;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Home-brewed enzymes information</span><span style = "font-weight:bold;">:</span></div><div class = "text-block"><span style = "font-weight:bold;">BstLF </span><span>polymerase</span><span style = "font-weight:bold;">:</span></div><div class = "text-block"><span>expression and purification </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 = ":;">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 purification </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 = ":;">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;">Testing</span></div><div class = "text-block">We test the buffers on SARS-CoV-2 LAMP or RT-LAMP reactions with synthetic dsDNA or ssRNA respectively. </div><div class = "text-block">The general performance of Homemade buffer seems appropriate and no amplification was obtained for non-template controls (NTC).</div><div class = "text-block">To properly formulate the buffer we tested different KCl concentrations which is known to be a critical factor in the buffer.</div><div class = "text-block">For the home-brewed BstLF, we found that 50 mM KCl is the best concentration, displaying equal or better performance than the commercial buffer. No amplification was observed for 150 mM KCl formulation.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/c3ryhefe.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/c3r4hefe.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. Biotechniques</span><span style = ":;font-weight:bold;">69</span><span style = ":;">, (2020).</span></a></div></div>

[STEPS]
?. [Stock solutions]
DTT (dithiothreitol)To prepare 10 ml of 1 M solution, weigh 1.54 g of DTT powder and dissolve it completely in 80 mL of H2O. Make up the volume to 10 mL with H2O (it can be done in a 15 ml conical tube). Filter-sterilize the solution with a 0.22 μm filter, make 100 uL - 1 ml aliquots and store at -20°C.
?. [Stock solutions]
EDTA (ethylenediaminetetraacetic acid)To prepare 500 ml of a 500 mM solution:Weigh 93.06 g of EDTA disodium salt dihydrate powder (MW = 372.24 g/mol) and dissolve it in 400 mL of H2O. At this concentration it shouldn't dissolve completely until you up the pH.Adjust to pH 8.0 or 7.1 with concentrated NaOH. It would be needed ~25 uL of 10M NaOH. It should be added slowly in a magnetic stirrer letting the EDTA to dissolve and the pH to equilibrate.  Transfer the solution to a graduated cylinder and complete the volume to 500 ml with H2O.Filter-sterilize the solution with a 0.22 μm filter Store at room temperature or 4°C.
?. [BstLF and MMLV storage buffer]
Enzyme storage buffer was made based on NEB indicated Enzymes Storage Conditions.
DTT should be added as fresh as possible. Add DTT and Glycerol at the moment of assembling the 1X Buffer. Freeze and thaw cycles are not recommended for DTT solutions, always use a fresh aliquot.
?. [BstLF and MMLV storage buffer]
To prepare  of 1x Storage Buffer add: molecular grade H2O 10x Storage Buffer DTT 1M Glycerol 100%Mix properly (by vortex), use immediately or store at
1.5 mL
599 µl
150 µl
1.5 µl
500 µl
-20 °C
?. [Stock solutions]
GlycerolMake an aliquot and sterilize by autoclave.
?. [LAMP and RT-LAMP reaction buffer]
This (RT-)LAMP assay buffer was made based on NEB 1X Isothermal Amplification Buffer Pack. 10X Isothermal Amplification Buffer is as follow:
?. [LAMP and RT-LAMP reaction buffer]
Prepare the following stock solutions:
In the "Stocks Solutions" section you can find the preparation of each solution.
?. [Stock solutions]
Potassium chloride (KCl)To prepare 50 ml of a 1 M solution, weigh 9.3188 g of KCl powder and completely dissolve it in 40 mL of H2O. Make up the volume to 50 mL with H2O (it can be done in a 50 ml conical tube). Filter-sterilize the solution with a 0.22 μm filter and store at room temperature.
?. [Stock solutions]
Ammonium sulfate ([NH4]2SO4)To prepare 50 ml of a 1 M solution, weigh 6.6070 g of (NH4)2SO4 powder and completely dissolve it in 40 mL of H2O. Make up the volume to 50 mL with H2O (it can be done in a 50 ml conical tube). Filter-sterilize the solution with a 0.22 μm filter and store at room temperature.
?. [Stock solutions]
Magnesium sulfate (MgSO4)To prepare 50 ml of a 1 M solution, weigh 12.3240 g of MgSO4*7H20 powder and completely dissolve it in 40 mL of H2O. Make up the volume to 50 mL with H2O (it can be done in a 50 ml conical tube). Filter-sterilize the solution with a 0.22 μm filter and store at room temperature.
It is possible to use MgSO4 instead of heptahidated but it is more  hygroscopic and would need desiccating before weighing .
?. [Stock solutions]
Tris⋅HCl To prepare 250 ml of a 1 M solution: Weigh 30.2850 g of Trizma Base powder and completely dissolve it in 200 mL of H2O. It should be done in a magnetic stirrer.Adjust to pH 8.8 or 7.1 with concentrated HCl (2.5 M). It should be done in a properly calibrated pH-meter, with continuous stirr and giving enough time to let the pH to equilibrate. It is important to check the temperature with a thermometer and use a heater if necessary to reach a temperature solution of 25°C the whole time. Transfer the solution to a graduated cylinder and complete the volume to 250 ml with H2O.Filter-sterilize the solution with a 0.22 μm filterStore at room temperature or 4°C up to six months.
Temperature has a significant effect on the pH of Tris buffers. It decreases ~0.028 pH units per each °C increase.
?. [Stock solutions]
Hydrochloric acid (HCl)To prepare 50 ml of 2.5 M solution:Weight 4.55 g HCl and completely dissolve it in 40 mL of H2O, or add 10.25 mL HCl 37% (d = 1.19) solution to 40 ml H20.Make up the volume to 50 ml with H2O.
?. [Stock solutions]
Filters:To sterilize the solutions and the assembled buffer 0.22 μm syringe filters of MCE or Cellulose acetate membranes could be used.Specifically we used: BioFil MCE membrane0.22 μmEDLABCellulose acetate0.22 μm
?. [LAMP and RT-LAMP reaction buffer]
To prepare  of 10X Isothermal Amplification Buffer add: Molecular H20    1M Tris-HCl (pH 8.8 @ 25°C)  1M (NH4)2SO4 2.5M KCl 1M MgSO4 Tween® 20 Adjust with NaOH 1 M (~ )Make up the volume to  with H2O (~ )Make aliquots and store at .
50 mL
20 mL
10 mL
5 mL
10 mL
1 mL
0.5 mL
560 µl
50 mL
3 mL
It shouldn't be autoclaved.The autoclave is not recommended for Tween® 20, (NH4)2SO4 and MgSO4 solutions.
-20 °C
?. [BstLF and MMLV storage buffer]
To prepare of 10x Storage Buffer add:molecular grade H2O 1M Tris-HCl ph 7.1  KCl 2.5M EDTA 500 mM Triton® X-100Make up the volume to with H2O  (~)Make aliquots and store at
10 mL
6 mL
1 mL
2 mL
20 µl
100 µl
10 mL
880 µl
-20 °C
In the "Stocks Solutions" section you can find the preparation of each solution.