Nov 19, 2020
Version 2
Freeze-drying (Lyophilization) and manufacturing of Corona Detective assay V.2
- 1CRI (Center for research and interdisciplinarity) Paris;
- 2Hackuarium
- Coronavirus Method Development Community
- Reclone.org (The Reagent Collaboration Network)
External link: https://app.jogl.io/project/181
Protocol Citation: Guy Aidelberg, Rachel Aronoff 2020. Freeze-drying (Lyophilization) and manufacturing of Corona Detective assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bpv4mn8wVersion created by Guy Aidelberg
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 19, 2020
Last Modified: November 19, 2020
Protocol Integer ID: 44700
Keywords: LAMP, Open Science, Covid19, Sars-Cov2, RT-LAMP, Lyophilization , Freeze-drying
Abstract
A protocol for freeze-drying (Lyophilization) of CoronaDetective tests,
and more generally any QUASR RT-LAMP reaction.
The amounts here are for one 96 well standard PCR plate and for 20 µL reactions
This can be scaled for any amount and size.
Guidelines
The amounts here are for one standard 96 well PCR plate (12 experiments), and for 20 µL reactions
This can be scaled for any amount of plates at any reaction volume.
Materials
MATERIALS
Isothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
Magnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
Recombinant RNasin(R) RNase Inhibitor, 10,000uPromegaCatalog #N2515
Deoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
2019-nCoV_N_Positive ControlIntegrated DNA TechnologiesCatalog #10006625
Bst 2.0 Warm Start DNA Polymerase Glycerol-freeNew England BiolabsCatalog #M0402B
D-( )-Trehalose dihydrateSigma AldrichCatalog #T5251
WarmStart® RTx (Glycerol-Free)New England BiolabsCatalog #M0439B-HC1
Safety warnings
There is no biohazard risk from producing these freeze-dried tubes, but care must be taken to avoid any potential contamination with target sequences or RNAse.
Best practices should be followed (appropriate PPE, RNaseAway, etc.).
Before start
Make sure to have all needed primers and reagents in sufficient quantities.
Fluorescence-tagged primers and complementary quencher sequences are essential for QUASR detection.
Standard Enzymes usually come in 50% Glycerol, so as to be stable in the -20C freezer. The glycerol interferes with freeze-drying.
Make sure your enzymes are Glycerol-free and stored at -80C.
Prepare the Harvest Right Lyophilizer system (or similar) for its freeze-drying run (Clean and Make sure vacuum is pulling)
Other systems are possible, and robotics are useful for scaling.
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 100 millimolar (mM) for 200 µL add together
Primer 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/srep44778
For the NM SARS CoV 2 primer set:
Fam-FIP/BIP 16 micromolar (µM) - 32 µL each
LB/LF 8 micromolar (µM) - 16 µL each
F3/B3 2 micromolar (µM) - 4 µL each
Anti-FIP-Q 24 micromolar (µM) - 48 µL
48 µL DNAse/RNAse free water
For the RNAseP human internal control primer set:
FIP/BIP 16 micromolar (µM) - 32 µL each
LB/Hex-LF 8 micromolar (µM) - 16 µL each
F3/B3 2 micromolar (µM) - 4 µL each
Anti-LF-Q 12 micromolar (µM) 24 µL
72 µL DNAse/RNAse free water
FD QUASR RT-LAMP Mastermix: In a 2ml tube mix together
280 µL dNTPs 10 millimolar (mM) each dNTP +
200 µL of each 10x Primer Mix (NM and RNAseP From step 1.1)+
5.33 µL Glycerol free Bst 2.0 WarmStart® DNA Polymerase 120000 U/ml +
8 µL Glycerol Free WarmStart® RTx Reverse Transcriptase 75000 U/ml +
400 µL Trehalose 50 Mass Percent (for a 50% trehalose solution, mix 200mg trehalose with 400uL H20 and filter or autoclave) +
506.67 µL DNAse/RNAse free water
Vortex all or mix by pipetting up and down and then spindown.
Rehydration Buffer: Either now or at a later time make the rehydration buffer. For a whole plate, mix
200 µL 10X Isothermal Amplification Buffer +
100 µL Magnesium Sulfate (MgSO4) Solution 100 millimolar (mM) +
1300 µL DNAse/RNAse free water ,
store in a cool dark place or in a fridge (4 °C ) (stable for shipment)
Dispensing: In each well of a PCR plate place 16 µL of the Mastermix from step 2
A digital dispenser or liquid handling robot is useful here for larger scales
Controls: Optionally, add 4 µL of Internal controls (Such as IDT 2019-nCoV_N_Positive Control) to selected tubes and mark them as such.
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 ~-40 °C for a few hours
then turn on the vacuum (aiming for 500mtorr) for another couple of hours,
then slowly heating by 10 °C every hour with the vacuum still on.
Equipment
Scientific Freeze Dryer
NAME
lyophilizer
TYPE
Harvest Right
BRAND
HRFD-Med-Sci-EU
SKU
LINK
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)