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Team:EPF-Lausanne/Protocols/SDS-PAGE
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Revision as of 12:48, 21 October 2009
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.
