Polymerase Chain Reaction (PCR)
NUS iGEM
Published: 2023-09-26 DOI: 10.17504/protocols.io.261gedrndv47/v1
Abstract
This is the common Polymerase Chain Reaction (PCR) protocol followed by the 2023 NUS-Singapore iGEM team.
Before start
Steps
1.
Prepare a PCR tube.
2.
Thaw the KOD OneTM PCR Master Mix (Blue) from the -20°C fridge.
3.
For a 50μL PCR sample, add the following into a PCR tube (follow the sequence):
| A | B |
|---|---|
| KOD OneTM PCR Master Mix (Blue) | 25μL |
| Each Primer (2 primers in total) | 1.5μL |
| DNA Template | 1μL |
| DI Water | 21μL |
4.
Vortex the PCR tubes to mix well.
5.
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 (∞) |
6.
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.
