Aug 24, 2020
nCoV-2019 McGill RT Protocol, Lunascript
Forked from a private protocol
- 1McGill University;
- 2University of Birmingham
- McGill Genome Centre
Protocol Citation: Shu-Huang Chen, Sarah J Reiling, Josh Quick, Ioannis Ragoussis 2020. nCoV-2019 McGill RT Protocol, Lunascript. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgekjte
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: August 07, 2020
Last Modified: August 24, 2020
Protocol Integer ID: 40166
Keywords: Cov-19 Nanopore Sequencing, Cov-19 Illumina Sequencing,
Abstract
Artic nCoV-2019 McGill modified Lunascript Reverse Transcriptase sequencing protocol.
Materials
MATERIALS
Water, nuclease free
LunaScript RT SuperMix KitNew England BiolabsCatalog #
E3010L
cDNA preparation
cDNA preparation
Mix the following components in a 0.2 mL 8-strip tube;
Component Volume
LunaScript RT SuperMix (5x) 4 µL
Nuclease-free water 5 µL
Template RNA 11 µL
Total 20 µL
Note
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
Note
A mastermix should be made up in the mastermix cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Incubate the reaction as follows:
25 °C for 00:02:00
55 °C for 00:20:00
95 °C for 00:01:00
Place on ice for 00:01:00 or store cDNA at -20C