Clarke:Constructing vectors

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  1. Streak selective plate from glycerol stocks of E. coli that contain the vector plasmids and incubate overnight. Plates stored at 4 °C.
  2. Pick colony from the plate and put in LB + antibiotic overnight.
  3. Spin down cells, run miniprep kit to isolate plasmid DNA. Plasmids stored in buffer EB at -20 °C.
  4. Take 1 μg of plasmid DNA, digest with restriction enzymes to give sticky ends that match the insert. If enzymes are heat inactivated, can store at -20 °C. (?)
  5. Separate vector from cutout by gel electrophoresis. Gel slice stored at 4 °C.
  6. Use gel extraction kit to recover vector. Vector stored in buffer EB at -20 °C.