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Sensor: Week 10 July 18-22
From 2011.igem.org
Contents |
Monday, July 18
The sensor group amplified the following constructs through the PCR protocol using the reverse primer:
| B0030+C0051 |
| B0031+C0051 |
| B0034+C0051 |
Tuesday, July 19
| Protocol | Part Involved in Protocol | |
|---|---|---|
| Restriction Digest | Inserts using EcoRI and SpeI: | B0030 + C0051 (R) B0031 + C0051 (R) B0034 + C0051 (R) Or B0034 |
| Vectors using EcoRI and XbaI: | Or34mcherry 34Cro Tev | |
| Ligation | B0030 + Or34mcherry B0031 + Or34mcherry B0034 + Or34mcherry Or + 34Cro B0034 + Tev | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. | |
Wednesday, July 20
The sensor group amplified the colonies that started growing on Tuesday, July 19 through colony PCR. The PCR products were run on an agarose gel, which indicated that all of the assemblies worked. The colonies were grown up in cultures.
Thursday, July 21
None of the cultures from Wednesday, July 20 grew any cells. The colonies from Tuesday, July 19 were grown again in culture in order for the plasmids to be extracted and sequenced.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Insert using XbaI and PstI: | OR+K648000 |
| Vectors using SpeI and PstI: | J23113 + B0031 + C0051 J23113 + B0034 + C0051 J23113 + B0030 + C0051 | |
| Ligation | J23113 + B0030 + C0051 + Or + K648000 J23113 + B0031 + C0051 + Or + K648000 J23113 + B0034 + C0051 + Or + K648000 | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. | |
Results: The above assembly attempts all yielded negative sequencing results, so the assembly process had to be repeated for the above constructs.
The following constructs were created using the ordered oligonucleotides and PCR:
| B0030 + C0051 |
| B0031 + C0051 |
| B0034 + C0051 |
Friday, July 22
The plasmids created on Tuesday, July 19, were extracted using the Omega Bio-Tek miniprep kit and sent to sequencing. The results showed that none of the assemblies worked, and would thus have to be repeated.
