05/08/13

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Isolation of ligated plasmid

  • The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
  • Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
  • there were 6 tubes containing the plasmid.
  • The plasmid concentration in each of the 6 tubes were measured using nanodrop.
  • 200ng of plasmid from each of the 6 different tube was used for digestion.

Digestion of ligated plasmid

  • Master Mix (changes were made from the protocol)
    • 2ul of NEB Buffer
    • 1ul of EcoRI
    • 1ul of PstI
    • varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
    • Final volume of each of the 6 tubes was 20ul.

Gel Electrophoresis

  • The product of digestion was run on agarose gel
    • This was done in order to confirm the presence of the limonene biobrick