Sep 26, 2023
Polymerase Chain Reaction (PCR)
- 1National University of Singapore
Protocol Citation: NUS iGEM 2023. Polymerase Chain Reaction (PCR). protocols.io https://dx.doi.org/10.17504/protocols.io.261gedrndv47/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: September 12, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 87654
Keywords: PCR, Polymerase Chain Reaction, KOD OneTM PCR master Mix, iGEM
Abstract
This is the common Polymerase Chain Reaction (PCR) protocol followed by the 2023 NUS-Singapore iGEM team.
Materials
- KOD OneTM PCR Master Mix (Blue)
- DNA Template
- Primer(s)
- DI Water
Safety warnings
Proper lab PPE must be worn.
Prepare a PCR tube.
Thaw the KOD OneTM PCR Master Mix (Blue) from the -20°C fridge.
For a 50μL PCR sample, add the following into a PCR tube (follow the sequence):
| Item | Volume | |
| KOD OneTM PCR Master Mix (Blue) | 25μL | |
| Each Primer (2 primers in total) | 1.5μL | |
| DNA Template | 1μL | |
| DI Water | 21μL |
Vortex the PCR tubes to mix well.
Place the PCR tube into the Thermal Cycler with the following conditions:
**Lid Temperature: 105°C; Volume: 50μL
| A | B | C | |
| 98°C | 10s | ||
| 68°C | 5s | Edit: -1°C per cycle | |
| 68°C | 1 minute | ||
| Go to step 1, repeat the cycle 13 times | |||
| 98°C | 10s | ||
| 55°C | 5s | ||
| 68°C | 1 minute | ||
| Go to step 5, repeat the cycle 25 times | |||
| 68°C | 5 minutes | ||
| 12°C | Infinite Loop (∞) | ||
Upon finishing, cancel the current protocol and take out the PCR sample from the Thermal Cycler. Keep the sample in the 4°C fridge for storage. Otherwise, gel electrophoresis may be carried out to isolate the DNA fragment of interest.