Team:EPF-Lausanne/Protocols/PCR with Taq Platinium

From 2009.igem.org

PCR with Taq Platinium Protocol



1. Add the following components to a sterile 0.5-ml microcentrifuge tube. Volumes are for a single 50-μl reaction, and can be scaled as needed. Prepare a master mix of common components for multiple reactions.

Components Volume Final concentration
10X PCR Buffer, Minus Mg 2,5 μl 1X
10 mM dNTP mixture 1 μl 0.2 mM each
50 mM MgCl2 1.5 μl 1.5 mM
Primer mix (10 μM each) 1 μl 0.2 μM each
Template DNA ≥1 μl (as required)
Platinum® Taq DNA Polymerase 0.2 μl 1.0 unit*
Autoclaved, distilled water to 25 μl Not applicable

*1.0 unit is sufficient for amplifying most targets. In some cases, more enzyme may be required (up to 2.5 units).


2. Cap the tubes, mix, and centrifuge briefly to collect the contents.

3. Incubate tubes in a thermal cycler at 94°C for 30 seconds to 2 minutes to completely denature the template and activate the enzyme.

4. Perform 25–35 cycles of PCR amplification as follows:

Denature 94°C for 30 seconds
Anneal 55°C for 30 seconds
Extend 72°C for 1 minutes per kb


5. Maintain the reaction at 4°C after cycling. The samples can be stored at –20°C until use. Analyze the products by agarose gel electrophoresis.