Colony PCR Protocol contributed by Lynn Hancock 1. Choose a reasonable sized colony (2-3 mm in diameter) and resuspend 100 µl ddH2O. 2. For PCR, 1 µl of colony suspension 4 µl 10 X PCR Buffer (without Mg++) 4 µl 25 mM MgCl2 Final conc. = 2.5 mM 1 µl dNTP's (2.5 mM each) Final conc. = 62.5 µM 0.4 µl Primer 1 (10 mM stock) Final conc. = 100 nM 0.4 µl Primer 2 (10 mM stock) Final conc. = 100 nM 28.7 µl ddH2O 0.5 µl Taq polymerase Total reaction volume = 40 µl Overlay sample with sterile mineral oil. PCR Reaction Conditions 96°C 2 minutes 94°C 40 seconds 55°C 40 seconds 72°C 1 minute per kb expected product 29 more cycles (30 total) 72°C 10 minutes 4°C Hold End