DNA Sequencing

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Restriction

Ligation

  1. Combination of 50 ng of vector with a 2-fold molar excess of insert.
  2. Addition of an appropriate volume of T4 Buffer
  3. Addition of 0.5-1 µl T4 ligase
  4. Adjustment of volume to 20 µl with ddH2O
  5. Incubation over night at 16 °C
  6. Deactivation of ligase for 10 min at 65 °C

Chemical transformation

  1. Thawing of competent cells on ice
  2. Addition of 1 µL DNA
  3. Incubation on ice for 20 min
  4. Heat shock for 1 min at 42 °C
  5. Incubation on ice for 5 min
  6. Addition of 270 µL LB
  7. Incubation in thermoblock for 45 min (incubation time dependent on used vector!) at 37 °C and 300 rpm
  8. Smearing of cells on plates and incubation at 37°C (centrifuge it, discard supernatant, resuspend cells in rest-medium, 20 µL on plate)