05/08/13

From 2013.igem.org

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**varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
**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.
** Final volume of each of the 6 tubes was 20ul.
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==Gel Electrophoresis=
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*The product of digestion was run on agarose gel
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** This was done in order to confirm the presence of the limonene biobrick

Revision as of 14:02, 5 August 2013

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.
  • 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