Team:Washington/Notebook/colony PCR

Colony PCR

1. Prepare one sterile 0.6mL tube with the following reaction mixture for each colony you intend to pick.
   1. 5uL Qiagen Master Mix
   2. 1uL 40uM VF2
   3. 1uL 40uM VR
2. Prepare one sterile 0.6mL tube with 20uL sterile diH2O for each colony you intend to pick.
3. Pick colonies
   1. Pick a single colony using a micropipettor with sterile tip. The pippettor should be set to 3uL
   2. Aspirate colony into 20uL diH2O vigorously to transfer cells to diH2O
   3. Transfer 3uL of diH2O containing cells to reaction mixture set up in step 1
4. Run reaction
   1. 94C - 3min
   2. 94C - 30s
   3. 55C - 30s
   4. 72C - 1min / kb gene
   5. repeat 2 - 4 29x
   6. 72C - 10min
   7. 4C - forever