FAQ: What controls should be run to test the cells and DNA when using T3 DNA Ligase?

Note: Use the same DNA concentration for each control, typically 0.1 -1.0 ng per transformation.
  1. Transforming uncut vector in the competent cells checks cell viability and tests the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic.
  2. Linearized vector tests for background due to uncleaved plasmid. Should be <1% of the value obtained in 1.
  3. Cut and religated plasmid tests ligase activity and DNA end integrity. Should be near the value obtained in 1.
  4. Cut, phosphatased, religated plasmid tests background due to incomplete phosphatase treatment. Should be <1% of the value obtained in 1.
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