Colony PCR

Colony PCR is useful in determining whether or not a specific colony on a plate has a sequence you desire. Primers for the specific sequence should be used when preparing the reaction cocktail. Examples of primers: antibiotic resistance, or primers flanking a cloned region. 1. Select colonies to analyze and number them on the bottom side of the plate. 2. Begin PCR program (program specific for optimal annealing temperature of primers) thermal cycler and press PAUSE, allowing the block to heat to 95 degrees. 3. Mix Colony PCR cocktail per the following recipe:

Buffer (10X) DNTPs (2.5 mM each) Taq (5 units/µL) Forward Primer (20 µM) Reverse Primer (20 µM) Water +Colony TOTAL

3.0µL 3.0µL 0.3µL

0.5µL 0.5µL 22.7µL 30µL 4. Add the cocktail to the PCR plate on ice without adding the colony. 5. Place the plate in the thermal cycler. 6. Using the pipette tip, touch the side of the selected colony and stir the tip around in the cocktail in the well corresponding to that colony. By touching only the side of the colony you ensudre that the majority of it stays intact. This is important if you plan to use the colony further. 7. Do this for all colonies to be analyzed, cover the plate with microseal film and allow the reaction to proceed by pressing PAUSE again. 8. Analyze the products and prepare cultures for positive lines, grow cultures for at least 12 hours.