TITLE: FloodLAMP LAMP Assay Protocol v4.1 (0.5ml, Strips, Multichannel)
AUTHORS: Randy True

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>FloodLAMP uses </span><a href="https://www.medrxiv.org/content/10.1101/2020.04.23.20076877v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">an assay chemistry</span></a><span> developed by Brian Rabe and Prof. Connie Cepko at </span><a href="https://cepko.hms.harvard.edu" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">The Cepko Lab at Harvard Medical School</span></a><span>. "Rabe Cepko," as it's known, uses an ultra cheap, streamlined front end (sample inactivation and RNA purification+concentration). This protocol has been chosen by many groups, from pharmaceutical companies to researchers working in the developing world. The same aspects that make it ideal for Africa also make it ideal for America—it's cheap, works well, and can be implemented by any basic chemistry or biology lab.</span></div><div class = "text-block"><span>This assay was </span><a href="https://www.medrxiv.org/content/10.1101/2020.05.12.20095638v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">validated clinically</span></a><span> in May by researchers at Mass General Hospital. In addition, the key amplification reagent (</span><a href="https://www.neb.com/products/m1804-warmstart-colorimetric-lamp-2x-master-mix-with-udg#Product%20Information" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">NEB Colorimetric LAMP Master Mix</span></a><span>) is the same product used in </span><a href="https://www.color.com/lamp-eua-press-release" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">the FDA-approved test by Color Genomics</span></a><span>, who does about 5K tests per day in the SF Bay Area. </span></div><div class = "text-block">Our current preferred version incorporates a transfer of the nucleic acid bound silica in ethanol to the PCR tubes (strips or plates) where the LAMP reaction is carried out. The pellet resuspends easily in the 80% ethanol and—once in the PCR tubes—can be quickly dried on a heat block. Then they are ready for addition of the 1X LAMP Reaction Mix. We have used alternatives to this method, including an elution of the nucleic acid and also a resuspension of the pellet in water or 1X PBS, then addition to the LAMP reaction. With the LAMP Master Mix (NEB 1804), the assay uses 2 prepared solutions: a NaI Binding Solution and the Glass Milk (also called "prepared silica"). </div><div class = "text-block"><span>On our </span><a href="http://floodlamp.bio" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">website</span></a><span> are our protocols in worksheet form as we use in the lab. This and more information will be coming soon.</span></div></div>

[STEPS]
?. [Purification ( .5mL sample)]
Spike selected samples with Twist RNA
?. [Purification ( .5mL sample)]
For each 8 samples, Add 45uL of glass milk (GM) to 2.25mL of binding solution (BS), vortex glass milk before using, flicking tube to be sure that there is no pellet at the bottomand it's evenly mixed
?. [Purification ( .5mL sample)]
Add 255uL of BSGM to 500ul samples, while adding BSGM pipette up and down 3x at bottom, 3x in middle, draw from middle. Start 10min timer after last addition.
?. [Purification ( .5mL sample)]
Vortex samples 3sec, before adding to rocker or rotator. Alternatively shake or vortex every 2min.
?. [Purification ( .5mL sample)]
Spin down samples for 1min
?. [Express Wash and Transfer]
Pour the supernatant into a "sample supernatant" 50ml Falcon tube (or other waste)
?. [Express Wash and Transfer]
Gently add 900uL of 80% EtOH, do not disturb the pellet (predraw about 100ul of air so next aspirate removes all liquid)
?. [Express Wash and Transfer]
Aspirate all of the EtOH using the same tip and discard in "ethanol supernatant" 50ml Falcon tube (or other waste)
?. [Express Wash and Transfer]
Add 100uL 80% EtOH, resuspend the pellet and transfer to labeled PCR strip
?. [Express Wash and Transfer]
When all are in PCR strips, with P200 multichannel pipette, transfer 20uL from each to 2nd PCR strip. Cap all strips.
?. [Express Wash and Transfer]
Spin down strips for 1min
?. [Express Wash and Transfer]
Aspirate supernatant with 200uL multichannel pipette, hugging side of tubes away from pellet
?. [Express Wash and Transfer]
Heat strips on heat block set at 65C for 5-10 minutes (open covered with foil), the pellet should look chalky and not wet
?. [LAMP Reaction]
Make the ALL MasterMix, two separate tubes for R and AN primers
?. [LAMP Reaction]
For each strip of 8, add 66uL dH2O, 22uL of 10X Guanidine, vortex 3s
?. [LAMP Reaction]
With multichannel pipette, add 25uL of ALL MM to each of the pellet strips using the multichannel 200uL pipette (AN for 80% pellet strip and R for 20%), pipet up and down 5x
?. [LAMP Reaction]
Add 22uL of the 10x primers (AN for 80% pellet strip and R for 20%), vortex 3s
?. [LAMP Reaction]
Add 110uL of LAMP MM, vortex 3s
?. [LAMP Reaction]
Cap strip tubes,  remove from holder, check caps, flick to mix, snap down
?. [LAMP Reaction]
Incubate strip tubes on heat block at 65C for 25min (can check at 20min and 30min)
?. [LAMP Reaction]
Remove strips, let cool for at least 1min before snapping photo.
?. [LAMP Reaction]
Add 25uL x n strips + 2uL (27, 52, ...) of the ALL MM to each tube in a new PCR strip, one strip for R and one for AN
?. [LAMP Reaction]
Observe tube color, should be bright pink. Note if orange tinted (likely pellet was not dry).