PCR Amplification (50 ul)

1. 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
2. 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

PCR Purification We used the QIAquick PCR Purification Kit from Qiagen.

1. Add 5 volumes of Buffer PB to 1 volume of PCR sample and mix. Apply sample to a spin column and centrifuge at 13,000 rpm for 30-60 seconds.
2. Discard flow-through.
3. Place the column back into the same tube.
4. Wash with 0.75 mL Buffer PE and centrifuge at 13,000 rpm for 30-60 seconds.
5. Discard flow-through and place the column back into the same tube.
6. Dry spin for an additional 1 minute at 13,000 rpm.
7. Place column in a clean 1.5 mL microcentrifuge tube. Add 50 uL of nuclease free water to the center of the membrane.
8. Let sit at room tempature for 10 minutes.
9. Centrifuge the column at 13,000 rpm for one minute to elute.
10. Store at -20°C.