| Colony PCR for 4 reactions | |
| 1. Thaw all reagents | |
| 2. Prepare a 10 μM primer mix. In a sterile 1.5 mL Eppendorf tube, add the following: | |
| a. 16 μL sterile H₂O | |
| b. 2 μL Forward Primer (from 100 μM stock) | |
| c. 2 μL Reverse Primer (from 100 μM stock) | |
| 3. Prepare the PCR mix. In a new sterile 1.5 mL Eppendorf tube, combine the following: | |
| a. 24 μL sterile H₂O | |
| b. 6 μL of the primer mix (prepared above) | |
| c. 30 μL of 2x PCR Master Mix | |
| 4. Aliquot 10 uL PCR mix into each well of 8-strip PCR tube. | |
| 5. Using a 10 uL pipette tip, pick a single colony from the LB agar plate. | |
| 6. Dip the pipette tip (with the picked colony) into a sterile 1.5 mL Eppendorf tube containing 200 μL of LB broth with the appropriate antibiotic. | |
| 7. Without changing the tip, immediately dispense the pipette tip into the colony PCR mix in the 8-strip PCR tube | |
| 8. after all colonies are picked, use channel pipette to gently dispense the colony and discard tips. | |
| 8. Place the tube into the thermocycler. | |