Team:Gaston Day School/Notebook
Notebook
On this page, Gaston Day School iGEM team would like to share with you our notes during the experiments from 09/01/2017 to 10/23/2017. The team leader Heena Saqib records the data patiently.
Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA
Methods:
For a 25 µL reaction
- In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity - Gently mix the reaction
- If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
- Transfer the PCR tube from ice to a PCR machine to begin thermocycling
For a 50 µL reaction
- In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity - Gently mix the reaction
- If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
- Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling
Thermocycling
The PCR machine should be set to run the following steps:
| Step | Temperature (°C) | Time |
|---|---|---|
| Initial denaturation | 98 | 30 seconds |
| 25-35 cycles | 98 (denaturation) 45-72 (annealing) see Note 1 72 (extension) |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
| Final extension | 72 | 2-5 minutes |
| Hold | 4 | Indefinitely |
Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/
