Sep 09, 2025
artic-measles/400/v1.0.0 Protocol
- Chris Kent1,2,
- Dominika Stepniak1,2
- 1ARTIC Network;
- 2University of Birmingham
- ARTIC
Protocol Citation: Chris Kent, Dominika Stepniak 2025. artic-measles/400/v1.0.0 Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvody39g4o/v1
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: July 23, 2025
Last Modified: September 09, 2025
Protocol Integer ID: 223064
Keywords: cdna generation via lunascript rt, cdna generation, primer pool, artic, artic locost amplicon, pcr reaction, downstream library preparation
Funders Acknowledgements:
Wellcome Trust
Grant ID: 313694/Z/24/Z
Disclaimer
For research use only.
Abstract
This protocol is how to generate the primer pools for artic-measles/400/v1.0.0. Then generate cDNA generation via LunaScript RT, and run the PCR reactions. Downstream library preparation relies on the ARTIC LoCost amplicon sequencing (SQK-NBD114).
Materials
Reagents:
| Component | Supplier | Part Number | |
| LunaScript® RT SuperMix Kit | NEB | M3010L | |
| Tris-EDTA (TE) | |||
| Nuclease-free water | |||
| Q5® Hot Start High-Fidelity 2X Master Mix | NEB | M0494S |
Equipment:
- Microcentrifuge
- Vortex mixer
- Thermal cycler (Ideally one for prePCR and one for PCR)
- P1000, P200, P20 and P2 pipette
- Ice bucket
- Timer
- Qubit fluorometer or equivalent
Troubleshooting
cDNA Synthesis
30m
Note
A positive control can also be included which may be a synthetic RNA constructs or high-titre clinical sample which can be diluted. This can help monitor run performance.
Mix the following components in a PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid.
| A | B | |
| Component | Volume | |
| LunaScript RT SuperMix (5X) | 2 μL | |
| Template RNA | 8 μL | |
| Total | 10 μL |
Components for RT reaction
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
To prevent pre-PCR contamination the mastermix should be added to the PCR strip-tubes/plate in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
RNA samples should be added in the extraction/sample addition cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Incubate the reaction as follows:
25 °C for 00:02:00
55 °C for 00:10:00
95 °C for 00:01:00
Hold at 4 °C
Primer pool preparation
1h
If making up primer pools from individual oligonucleotides (oligos)
Note
If using pre-pooled scheme. Please skip to Step 7
Briefly spin down lyophilised oligos to ensure pellet is at bottom of the tube.
Resuspend oligos in 1x Tris-EDTA (TE) to a of concentration 100 micromolar (µM) , vortex thoroughly and spin down.
Dilute the 100 micromolar (µM) pools 1:10 in molecular grade water, to generate 10 micromolar (µM) working primer stocks.
Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a
1.5 mL Eppendorf tube labelled "Pool 1 100 micromolar (µM) ". Repeat the process for all even region primers for Pool 2. These are your 100 micromolar (µM) stocks of each primer pool.
Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
Note
For artic-measles/400/v1.0.0; there are 193 primers in Pool 1 and 181 primers in Pool2. Therefore, to ensure each primer is at ~15 nanomolar (nM) use either; 7.3 µL of 10 micromolar (µM) working primer stocks or 0.73 µL of 100 micromolar (µM) primer stocks per 25 µL PCR reaction.
Multiplex PCR
4h
Add 2.5 µL cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Note
cDNA should be added in the extraction and sample addition cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
| A | B | C | |
| Component | Reaction 1 | Reaction 2 | |
| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 μL | 12.5 μL | |
| Pool 1 (10μM) | 7.3 μL | 0 μL | |
| Pool 2 (10μM) | 0 μL | 7.3 μL | |
| Nuclease-free water | 2.7 μL | 2.7 μL | |
| Total | 22.5 μL | 22.5 μL |
Note
To prevent pre-PCR contamination the mastermix for each pool should be made up in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use and aliquoted into PCR strip-tubes/plate
Set-up the following program on the thermal cycler:
Step Temperature Time Cycles
Heat Activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 25-35
Annealing 65 °C 00:05:00 25-35
Hold 4 °C Indefinite 1
30s
Quality Control
The methods of quality control are very dependant on what is available in the lab. You want to check PCR yield and product size.
Yield can be measured on NanoDrop, Qubits or other Fluorometers. Yields are typically between 10 ng/uL to 100 ng/uL .
For Qubit, see step 25 in linked protocol under Library Preparation.
For product size, electrophoresis gels or TapeStations can be used to measure. A single band should be present at the size of the amplicons (~400bp). Some smear at ~20-60bp is expected (due to primers) particularly in negatives and low input samples.
Library preparation
