PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
Yin-Tse Huang
Published: 2022-04-24 DOI: 10.17504/protocols.io.81wgb644qlpk/v2
Abstract
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
Steps
For generic primers
1.
PCR mixture PCR condition for generic primers
| A | B | C |
|---|---|---|
| Component | Volume | Final conc. |
| Template (< 1 µg) | X µL | - |
| Forward Primer (10 µM) | 1 µL | 0.4 µM |
| Reverse Primer (10 µM) | 1 µL | 0.4 µM |
| 2x Master Mix | 12.5 µL | - |
| ddH20 | up to 25 µL | - |
| Total volume | 25 µL | - |
2.
PCR condition for generic primers
| A | B | C | D |
|---|---|---|---|
| Step | Temp | Sec | Cycle |
| Initial denaturation | 95 ºC | 180 | |
| Denaturation | 95 ºC | 30 | 35 cycles |
| Annealing | 53-58 ºC | 30 | |
| Extension | 72 ºC | 60 | |
| Final extension | 72 ºC | 210 | |
| Preservation | 4 ºC | ∞ |
For barcoded primers
3.
PCR mixture for barcoded primers
| A | B | C |
|---|---|---|
| Component | Volume | Final conc. |
| Template (< 1 µg) | X µL | - |
| Forward Primer (10 µM) | 5 µL | 2 µM |
| Reverse Primer (10 µM) | 5 µL | 2 µM |
| 2x Master Mix | 12.5 µL | - |
| ddH20 | up to 25 µL | - |
| Total volume | 25 µL | - |
4.
PCR condition for barcoded primers
| A | B | C | D |
|---|---|---|---|
| Step | Temp | Sec | Cycle |
| Initial denaturation | 95 ºC | 180 | |
| Denaturation | 98 ºC | 20 | 35 cycles |
| Annealing | 64 ºC | ||
| Extension | 72 ºC | ||
| Final extension | 72 ºC | 210 | |
| Preservation | Preservation | 4 ºC | ∞ |
