Team:TU Delft/protocols/PCR
From 2010.igem.org
PCR
Materials:
- Taq PCR Master Mix (Qiagen) is premixed solution containing Taq DNA Polymerase, PCR Buffer and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 μM each dNTP.
- Primer solutions 5 mol/mL
- Template DNA (plasmid at 50 pg – 1 ng/μL)
Protocol:
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice. Preparation of reaction mixture:
1. Gently vortex and briefly centrifuge all solutions after thawing
2. Keep solutions on ice
3. Add to a thin walled PCR tube, on ice, for a 100 μL reaction:
1× pre-mix
Component | Sample |
Taq PCR Master Mix (Qiagen) | 25 μL |
Primer 1 | 3 μL |
Primer 2 | 3 μL |
DNA template | 1 μL |
H20 | 18 μL |
4. Gently vortex the sample and briefly centrifuge (5 sec) to collect all droplets at the bottom of the tube
5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.
PCR program:
Step | Annealing Temperature | Time, min:sec | Number of cycles |
Initial denaturation | 94 °C | 2:00 | 1 |
Denaturation | 94 °C | 1:00 | 30 |
Annealing | x °C * | 0:45 | 30 |
Extension | 72 °C | 1:00 | 30 |
Final Extension | 72 °C | 5:00 | 1 |
* Calculate the optimal annealing temperature = 3x G/C + 2x A/T