from Rob Holdcraft- Ann Rougvie Lab
This protocol allows rapid detection of transformation success when primers are available to allow determination of correct ligation products by size or hybridization.
Start => Bacterial colonies from transformation.
- Draw grid on clean LB-amp or LB-carb plate (~28 sectors).
- 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
- Heat at 95 degrees for 10 minutes. ** This can be done in PCR machine.
- 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.
- 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