IGEM:Paris Bettencourt 2012/Protocols/RCR Taq

1. Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
2. 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

3. Gently vortex the samples and spin down.
4. When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
5. 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

• Comments: LID = 100°C