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 (40 preps)

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