From 2013.igem.org

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions


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.
samplevolume(ul)concentration (ng/ul)yield(ug)

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

IGem Digested 050813.jpg

  • The lanes are in the following order:
    • Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
  • We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1,10.2 and 10.3. To further determine the composition of samples, we did a single digest on the samples.

Single digest

  • For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
  • The samples are run on 0.8% agarose gel.