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Bacterial Colony PCR

from Rob Holdcraft- Ann Rougvie Lab


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. Draw grid on clean LB-amp or LB-carb plate (~28 sectors).
  2. Select colonies to pick. Streak portion of colony to numbered sector and place the remainder in a correspondingly numbered PCR tube with 50uL of lysis buffer
  3. Heat at 95 degrees for 10 minutes. ** This can be done in PCR machine.
  4. Spin lysis solution on high for 10 minutes to pellet cellular debris, and remove 4uL for PCR. ** PCR design depends on primers and analysis of product, etc.

Reagents/Solutions

Lysis Buffer

  • 1% Triton X-100
  • 20mM Tris*Cl pH 8.0
  • 2mM EDTA pH 8.0

For 2mLs (40preps)

  • 1.75 mL H2O
  • 200uL 10% Triton X-100
  • 40uL 1M Tris*Cl pH 8.0
  • 8uL 0.5M EDTA pH 8.0