Colony PCR

PCR can be performed directly from a scraped cell colony, using an extended denaturing step to cause lysis of the cells. This is a nice time saving protocol for PCRing targets directly from antibiotic selective plate media.

Colony PCR with Patch Plating 1. If subculturing via patch plate, divide a warm LB-Amp plate into a grid (commonly 3x3), labelling each square on the back of the plate.

  1. Using a sterile wooden stick, aseptically pick up a transformed colony and smear some of the colony in a PCR tube.

  2. If subculturing via patch plate, use the same stick to patch it on one of the squares.

  3. Repeat steps 2 and 3 for multiple colonies.

  4. Place patch plate in 37oC incubator. Move to a 4oC fridge the next day.

  5. Add reagents to the PCR tubes as described in table 1. The reagent volumes can be modified to fit whatever your typical PCR setup looks like.

  6. Run the PCR cycles as detailed in table 2.

  7. The resultant PCR products can be run through a gel for gel extraction or cleaned with a PCR cleanup kit.

Figure 1 displays the results of the colony PCR using all 6 patched colonies. Lanes 1 - 6 contain the colony PCR products of 6 transformed colonies. A 1 kb ladder was loaded into lane 7. Lanes 8 and 9 were PCR products of untransformed control colonies.


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