Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner

1. Add 5 ml liquid LB media into 12 ml culture tubes
2. Add 5 μl of appropriate antibiotic into the broth
3. Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
4. Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm

Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA

Methods:
For a 25 µL reaction

1. 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
2. Gently mix the reaction
3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
4. Transfer the PCR tube from ice to a PCR machine to begin thermocycling

For a 50 µL reaction

1. 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
2. Gently mix the reaction
3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
4. 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:

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/

Materials:
Sterile Water
25 µL RedTaq mastermix
1 E. coli colony
2.5 µL of 10 µM forward primer
2.5 µL of 10 µM reverse primer

Methods:

1. Add a single colony of cells to 50 µL of water. Incubate at 95C for a minute to lyse the cells.
2. Combine 1 µL cell lysate, 25 µL RedTaq mastermix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and sterile water up to 50 µL.
   Note: It is important to add RedTaq Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
3. Incubate in the thermocycler - Taq has a lower optimum temperature than Phusion.

Thermocycling
The PCR machine should be set to run the following steps:

Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.