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Bacterial Colony PCR

Bacterial Colony PCR


Objective:

This protocol allows rapid detection of transformation success when primers are available to allow determination of correct ligation products by size or hybridization.

Procedures:

    Start => Bacterial colonies from transformation.
  1. Prepare 2mL culture tubes w/ an appropriate selection media for your plasmid of interest, label 1.5mL tubes and place inside culture tube as a cap.
  2. Prepare PCR master mix: buffer, MgCl2, dNTPs, primers, sucrose red, and enzyme- Aliquot 25µL of master mix into a PCR tube for each colony to be picked. (See general PCR instructions for more instructions on how to set up the PCR reaction.)
  3. Select colonies to pick. Pick colony with a sterile toothpick or pipet tip. Dab the toothpick or pipet tip into the master mix then place the toothpick or pipet tip in a correspondingly labeled culture tube.
  4. Run PCR w/ appropriate conditions for your primers and expected product, Making sure to begin your PCR protocol with an extended time at 95°, (e.g. 5 minutes).
  5. Grow the 2mL cultures O/N at 37°.
  6. Run 10µL of the PCR reaction on an agarose gel to identify which cultures to keep for plasmid DNA isolation.

Direct questions or comments concerning this web page to Karl Clark: kclark@biosci.cbs.umn.edu
Last modified July 12, 2000
URL = http://biosci.cbs.umn.edu/~kclark/protocols/bactPCR.html%3CBR>
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