Mar 25, 2024

Public workspacebHEV genotpying RT-PCR V.1

This protocol is a draft, published without a DOI.
  • 1University of Hong Kong
Siu Fung Ho
Open access
Protocol Citation: Siu Fung Ho 2024. bHEV genotpying RT-PCR. protocols.io https://protocols.io/view/bhev-genotpying-rt-pcr-dabx2apn
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: March 08, 2024
Last Modified: March 25, 2024
Protocol Integer ID: 96343
Abstract
bHEV genotpying RT-PCR
Materials
  • Superscript IV Reverse Transcriptase
  • 2X Plus High Fidelity Fast PCR Master Mix
  • Genotyping Primers
  • Nuclease-free water
  • Template RNA
  • TBE Buffer
  • Agarose
  • 1 Kb ladder/Loading Dye
Reverse Transcription
Reverse Transcription
Pre-warm the 5x SSIV buffer to RT
Make up the primer annealing master mix in a 2ml Eppendorf. Per sample volumes:

Component Volume
dNTPs 10mM Amount1 µL
Reverse Primer 2mM Amount1 µL
Nuclease-free water Up to Amount13 µL

DivideAmount13 µL of master mix between reaction tubes.

Add up to Amount5 µg RNA template to reaction tubes.

Mix and briefly centrifuge.
Heat the RNA-primer mix at 65°C for 5 minutes.
Incubate on ice for at least 1 minute.
Vortex and briefly centrifuge the 5x SSIV Buffer.
Make up the RT master mix in a 2ml Eppendorf. Per reaction volumes:

Component Volume
5x SSIV Buffer Amount4 µL
DTT 100 μM Amount1 µL
RNase Inhibitor Amount1 µL
Reverse Transcriptase (200 U/μL) Amount1 µL

Note
Mastermix should be made up and aliquoted into PCR tubes in a mastermix cabinet. Cabinet and tubes should be cleaned with decontamination wipes/70% ethanol and UV sterilised before and after use.

Mix and briefly centrifuge RT master mix
Add Amount7 µL of RT master mix to annealed RNA

Note
This should be done in the pre-PCR template addition room.


Incubate the combined reaction mixture at 50°C for 10 minutes
Inactivate the reaction by incubating it at 80°C for 10 minutes. During this incubation, PCR mastermix can be setup (next step).
Polyermase Chain Reaction
Polyermase Chain Reaction
18m
Setup mastermix in a 1.5 mL eppendorf tube. The following amounts are for a single reaction

Component Volume
2x Reaction Mix Amount25 µL
Forward Primer 10 μM Amount2 µL
Reverse Primer 10 μM Amount2 µL
Nuclease-free water Amount16 µL
Total Amount50 µL

Note
Mastermix should be made up and aliquoted into PCR tubes in a mastermix cabinet. Cabinet and tubes should be cleaned with decontamination wipes/70% ethanol and UV sterilised before and after use.

Add Amount5 µL template cDNA to the PCR tubes.

Note
This should be done in the pre-PCR template addition room.

Gently mix contents of each tube by pipetting and spin down.
Program the following PCR cycles into the thermal cycler.

Step Temperature Time Cycles
Reverse transcription Temperature50 °C Duration00:10:00 1
Inactivation Temperature98 °C Duration00:02:00 1
Denaturation Temperature98 °C Duration00:00:10 10
Annealing Temperature50 °C Duration00:00:10 10
Extension Temperature72 °C Duration00:00:30 10
Denaturation Temperature98 °C Duration00:00:10 30
Annealing Temperature60 °C Duration00:00:10 30
Extension Temperature72 °C Duration00:00:30 30
Final extension Temperature72 °C Duration00:05:00 1

Protocol references