- Sterile 0.6mL plastic tubes
- LB-agar plate with appropriate antibiotic
- Oligonucleotide pair (usually VF2 and VR)
- PCR supermix
- PCR machine
- Sterile pipet tips
- Prepare one sterile 0.6mL tube with 20μL ddH2O for each colony you intend to pick.
- Prepare LB-agar plate with appropriate antibiotic to use as index plate.
- Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3μL
- Inoculate tip with colony into tube. Pipet up and down to ensure cells are transferred to tube. Pipet 3μL of cells suspended in water onto index plate.
- Repeat for as many colonies as you intend to pick.
- 9 μL PCR supermix
- 0.25 μL 40nM VF2
- 0.25 μL 40nM VR
- 0.5 μL colony template
- 95°C for 15 mins
- 94°C for 30 secs
- 56°C for 30 secs
- 68°C for 1 min per kb of expected product
- Repeat 2-4 39 times.
- 68°C for 20 mins
- 4°C forever
Run a gel to determine amplification product length.