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05/08/13
From 2013.igem.org
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Contents |
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.
| sample | volume(ul) | concentration (ng/ul) | yield(ug) |
| 5.1 | 99 | 75 | 7.4 |
| 5.2 | 100 | 44.2 | 4.4 |
| 5.3 | 98 | 46.7 | 4.6 |
| 10.1 | 46 | 61.8 | 2.84 |
| 10.2 | 44 | 77.1 | 3.39 |
| 10.3 | 47 | 13.7 | 0.64 |
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
- 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.
