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Crimson Taq DNA Polymerase with (Mg-free) Buffer Protocol |  | | Crimson Taq DNA Polymerase with (Mg-free) Buffer Protocol |
Protocol
- Add the following components to a thin-walled PCR tube on ice:
| Component | 25 µl reaction | 50 µl reaction | Final Conc. | | 5X Crimson Taq (Mg-free) Reaction Buffer | 5 µl | 10 µl | 1X | | 10 mM dNTP | 0.5 µl | 1 µl | 200 µM | | 10 µM Forward Primer | 0.5 µl | 1 µl | 0.2 µM (0.05–1 µM) | | 10 µM Reverse Primer | 0.5 µl | 1 µl | 0.2 µM (0.05–1 µM) | | 25 mM MgCl2 | 1.5 µl | 3 µl | 1.5 mM | | Template DNA | variable | variable | <1000 ng | | Crimson Taq DNA Polymerase | 0.125 µl | 0.25 µl | 1.25 units/50 µl PCR | | Nuclease-free water | to 25 µl | to 50 µl | |
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without heated lid. - Transfer PCR tubes to a PCR machine with the block preheated to 94-95°C and begin the programmed cycling:
Initial denaturation: 95°C 30 seconds
25-45 cycles: 95°C 20 seconds; 50-68°C 15-60 seconds; 68°C 1 minute per kb
Final extension: 68°C 5 minutes
Final soak: 4-10°C
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