Sep 01, 2020
Mmolecular COVID-19 Extraction-Free Direct-One-Step Fast Cycling Protocol
- 1Mmolecular.com
- Coronavirus Method Development Community
- Mmolecular
Protocol Citation: steve.puts 2020. Mmolecular COVID-19 Extraction-Free Direct-One-Step Fast Cycling Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkjpkumn
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: In development
We are still developing and optimizing this protocol.
Created: September 01, 2020
Last Modified: September 01, 2020
Protocol Integer ID: 41295
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Abstract
The Direct One-Step RT-qPCR COVID-19 Test Kit is designed for quantitative
real-time analysis of target RNA directly from, swabs without the requirement of any prior
RNA purification steps.
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
Additional Storage Conditions: avoid freeze/thaw cycles stable at 4 °C for up to 4 weeks
Shelf Life: 12 months
Form: liquid / lyophilized
Well plate preparation
Well plate preparation
5m
5m
Pre-mix the reagents for 81 wells 81 µL total volume
40.5 µL Direct reaction mix
3.25 µL Enzyme mix
8.25 µL Primers and probes mix
29 µL PCR grade water
2m
Pipette the mix onto the 81 well plate.
To gain speed, use a pipette-ing robot.
3m
Sample processing
Sample processing
9m 30s
9m 30s
Place the swab brush into a 1.5ml microcentrifuge tube containing 135 µL PCR-grade water and 15 µL PBS (10x conc) .
Rotate the brush 5-10 times.
Squeeze the brush and remove it from the tube.
1m
Centrifuge at 12000 x g for 3 min at room temperature
3m
Discard the supernatant.
Add 4.5 µL PCR-grade water and 0.5 µL Extraction Buffer
Briefly flick the sample.
1m
Incubate for 3 min at room temperature for lysis and release of RNA.
3m
Centrifuge briefly and transfer 0.25 µL sample to the well plate.
Pipette up/down to make sure the sample has fully released from the pipette.
1m
Cover the sample with 1 µL mineral oil
30s
Cycling
Cycling
15m 30s
15m 30s
Load the well plate into the cycler and move the chamber door down.
Click run.
Run with parameters:
- RT 50C for 3 min
- Denaturation 95C for 0s (maximum speed)
- Annealing 60C for 0s (maximum speed)
15m 30s