To a small PCR tube (on ice) add:

• enough water to fill to 50 ul (19 uL)
• 1 ul of template DNA
• 2.5 ul each of 10 uM forward and reverse primers
• 25 ul of PCR Master Mix

Quickly transfer PCR tubes from ice to a preheated thermocycler with the following conditions:

• initial denaturation: 94°C for 30 seconds
• 25-35 cycles: 94°C for 15-30 seconds (we did 30 sec)
• 45-68°C for 15-60 seconds (we did 60°C for 30 sec)
• 68°C for 1min/kb (we did 2 min)
• final extension: 68°C for 5 min
• hold: 4°C for as long as necessary

NOTE: For colony PCR just add a single colony using a pipette tip as the last thing you add to the solution instead of the template DNA. Twirl the colony into the solution in the tube and then proceed as usual