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Star Republic: Guide for Biologists

PCR amplification of single clones

1. Transfer 5 ml overnight culture to a microcentrifuge tube or 96 well plate containing 95 ml of ddH2O. For clones on a LB plate, pick single clones using toothpicks, and resuspend cells in 100 ml of ddH2O.

2. Boil for 5 minutes or 95 C for 10 minutes if using 96 well plate to lysis cells.

3. Remove cell debris by brief centrifugation. For 96 well plate, centrifuge at 1200xg for 3 minutes.

4. Clones are amplified in 50 ml PCR reactions. Make a master mix, add 48 ml of master mix to 2 ml of DNA.

5. Run 5 ml on an argarose gel to check for amplifications.