TITLE: Freeze-drying (Lyophilization) and manufacturing of Corona Detective assay
AUTHORS: Guy Aidelberg, Rachel Aronoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for freeze-drying (Lyophilization) of CoronaDetective tests, </div><div class = "text-block">and more generally any QUASR RT-LAMP reaction.</div><div class = "text-block"><span>The amounts here are for one 96 well standard PCR plate and for </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span> reactions </span></div><div class = "text-block">This can be scaled for any amount and size. </div></div>

[STEPS]
?. Thaw components (dNTPs, 10X Primer mixes (need to be made), MgSO4,  Isothermal amplification buffer, and Enzymes)Vortex and quickly spin tubes down before opening for dispensing.This protocol is for one standard 96 well PCR plate and can be scaled as needed.
?. 10X Primer mix: assuming your primer stocks are at   for    add togetherPrimer sequences taken from: https://www.nature.com/articles/s41587-020-0513-4 (Supplementary Data 2)and adapted for QUASR detection according to https://www.nature.com/articles/srep44778For the NM SARS CoV 2 primer set: Fam-FIP/BIP   -   LB/LF  -    F3/B3 -       Anti-FIP-Q   - For the RNAseP human internal control primer set: FIP/BIP   -   LB/Hex-LF  -    F3/B3 -      Anti-LF-Q
200 µl
[each]
[each]
[each]
48 µl
[DNAse/RNAse free water]
[each]
[each]
[each]
24 µl
[DNAse/RNAse free water]
?. FD QUASR RT-LAMP Mastermix: In a 2ml tube mix together + (NM and RNAseP From step 1.1)++ + (for a 50% trehalose solution, mix 200mg trehalose with 400uL H20 and filter or autoclave) +Vortex all or mix by pipetting up and down and then spindown.
[dNTPs]
[each dNTP]
[ of each 10x Primer Mix]
[Glycerol free  Bst 2.0 WarmStart® DNA Polymerase]
[Glycerol Free WarmStart® RTx Reverse Transcriptase]
[Trehalose]
[DNAse/RNAse free water]
?. Rehydration Buffer: Either now or at a later time make the rehydration buffer. For a whole plate, mix + + , store in a cool dark place or in a fridge () (stable for shipment)
[10X Isothermal Amplification Buffer]
[Magnesium Sulfate (MgSO4) Solution]
[DNAse/RNAse free water]
4 °C
?. Dispensing: In each well of a PCR plate place   of the  Mastermix from step 2A digital dispenser or liquid handling robot is useful here for larger scales
16 µl
?. Controls: Optionally, add  of Internal controls (Such as IDT 2019-nCoV_N_Positive Control) to selected tubes and mark them as such.
4 µl
?. Sealing and piercing: Seal the plate(s) with either foil or parafilm and then make a small puncture in the seal of each tube. This is done when reactions are freeze-dried in order to prevent the small pellets from “jumping” out of the tubes under vacuum.  A 96-Pin Replicator is perfect for this but a multi-pipette, toothpick, tip, scalpel, or scissors will do.
?. Freeze-drying: Depending on your freeze drier, you might need to now freeze the tubes, and make sure they remain frozen (such as by placing in a frozen metal rack or touching a frozen metal block). Otherwise, simply place in a freeze-drier and run overnight or until done. We start by freezing to ~ for a few hoursthen turn on the vacuum (aiming for 500mtorr) for another couple of hours,then slowly heating by  every hour with the vacuum still on.
-40 °C
10 °C
?. Storage: Make sure each tube has a similarly sized dried pellet and reseal the plate with either film, foil, or caps. Store in a dark and dry place preferably in a sealed bag with a desiccant. (stable for shipment)