IGEM:Paris Bettencourt 2012/Protocols/RCR Taq
From OpenWetWare
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
| PCR Master Mix (2x)
| 25 ul
|
| Forward primer
| 0.1 - 1.0 uM
|
| Reverse primer
| 0.1 - 1.0 uM
|
| Template DNA
| 10 pg - 1 ug
|
| Water (Nuclease free)
| to 50 ul
|
| Total volume:
| 50 ul
|
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Step
| Temperature, °C
| Time
| Number of cycles
|
| Initial denaturation
| 95
| 10 min
| 1
|
| Denaturation
| 95
| 30 s
| 30
|
| Annealing
| 50
| 30 s
|
| Extension
| 72
| 1 min/kb
|
| Final Extension
| 72
| 10 min
| 1
|
| End
| 8
| Forever
| 1
|
