Sep 23, 2019
Q5 PCR DNA Amplificaiton (Protocol for Q5® High-Fidelity 2X Master Mix)
- Alba Balletbó1
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Alba Balletbó 2019. Q5 PCR DNA Amplificaiton (Protocol for Q5® High-Fidelity 2X Master Mix). protocols.io https://dx.doi.org/10.17504/protocols.io.7cwhixe
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: September 15, 2019
Last Modified: September 23, 2019
Protocol Integer ID: 27766
Keywords: q5 pcr dna amplificaiton, pcr, fidelity 2x master mix, protocol, dna
Abstract
This protocol is for PCR with Q5® High-Fidelity 2X Master Mix
Guidelines
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0492L
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
| Reagent | 25 µl Reaction | 50 µl Reaction | Final concentration | |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X | |
| Forward Primer (10 µM) | 1.25 µl | 2.5 µl | 0.5 µM | |
| Reverse Primer (10 µM) | 1.25 µl | 2.5 µl | 0.5 µM | |
| Template DNA | variable | variable | < 1,000 ng | |
| Nuclease-Free Water | to 25 µl | to 50 µl |
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Troubleshooting
Safety warnings
PCR reagents are classified as non-hazardous. Follow the specified handling and disposal considerations included in the safety data sheets provided by the manufacturer.
Before start
Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Add all components in a 250 μL tube making up to a 25 or 50 μl reaction. If performing various PCR with different templates, a Master Mix is recommended to be done.
When doing a Master Mix always add Q5 enzyme last, then vortex the solution briefly and centrifugate before use.
'Pro-Tip': Use DMSO 3% or 5X GC enhancer as PCR additives when amplifying particularly difficult or high GC amplicons.
Gently mix the PCR reactions and transfer the tubes to a thermocycler. Thermocycling conditions for a routine PCR:
| Step | Temperature | Time | |
| Initial Denaturation | 98°C | 30 seconds | |
| 25–35 Cycles | 98°C | 5–10 seconds | |
| *50–72°C | 10–30 seconds | ||
| 72°C | 20–30 seconds/kb | ||
| Final Extension | 72°C | 2 minutes | |
| Hold | 4–10°C |
*The use of the NEBTm Calculator is highly recommended.