Colony PCR

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  1. Mark a clean agar plate (with required antibiotic) with a grid; make sure top is clearly defined.
  2. Prepare PCR tubes containing 10μl PCR reaction mix/tube. Label tubes to correspond to grid coordinates. Place on ice.
  3. Pick a colony using a sterile loop, streak on one portion of the agar grid. Then swirl remaining bacteria into the equivalent PCR tube. KEEP ON ICE.
  4. Incubate plate overnight at 37°C to grow up colonies.
  5. Run PCR reaction at required temperatures, 30-35 cycles. Make sure first step of cycle is 94-96°C, 2 min, to lyse the bacteria.
  6. Run PCR reactions on gel to identify positive clones. Pick 4-5 positive clones per desired vector, conduct mini-prep, and send for sequencing.