05/08/13

From 2013.igem.org

(Difference between revisions)
(Gel Electrophoresis)
(Gel Electrophoresis)
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[[File:iGem_Digested_050813.jpg]]
[[File:iGem_Digested_050813.jpg]]
*The lanes are in the following order:
*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.
+
**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 and 10.2. To determine the composition of other samples, we are doing a single digest on the samples.

Revision as of 09:08, 6 August 2013

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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.
samplevolume(ul)concentration (ng/ul)yield(ug)
5.199757.4
5.210044.24.4
5.39846.74.6
10.14661.82.84
10.24477.13.39
10.34713.70.64

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 and 10.2. To determine the composition of other samples, we are doing a single digest on the samples.