Knight:Colony PCR

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Contents

Materials

  • Sterile 0.6mL plastic tubes
  • LB-agar plate with appropriate antibiotic
  • Oligonucleotide pair (usually VF2 and VR)
  • PCR supermix
  • PCR machine
  • Pipetman
  • Sterile pipet tips

Procedure

Picking colonies

  1. Prepare one sterile 0.6mL tube with 20μL ddH2O for each colony you intend to pick.
  2. Prepare LB-agar plate with appropriate antibiotic to use as index plate.
  3. Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3μL
  4. 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.
  5. Repeat for as many colonies as you intend to pick.

Reaction mixture

1X Reaction

  • 9 μL PCR supermix
  • 0.25 μL 40nM VF2
  • 0.25 μL 40nM VR
  • 0.5 μL colony template

PCR conditions

  1. 95°C for 15 mins
  2. 94°C for 30 secs
  3. 56°C for 30 secs
  4. 68°C for 1 min per kb of expected product
  5. Repeat 2-4 39 times.
  6. 68°C for 20 mins
  7. 4°C forever

Run a gel to determine amplification product length.

Notes

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