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- Mark a clean agar plate (with required antibiotic) with a grid; make sure top is clearly defined.
- Prepare PCR tubes containing 10μl PCR reaction mix/tube. Label tubes to correspond to grid coordinates. Place on ice.
- 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.
- Incubate plate overnight at 37°C to grow up colonies.
- 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.
- Run PCR reactions on gel to identify positive clones. Pick 4-5 positive clones per desired vector, conduct mini-prep, and send for sequencing.