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Team:Yale/Our Project/Notebook/Week 3
From 2010.igem.org
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<li>Samples 2,4, and 7 were too dilute to be of use, but ran 50 uL digestions of the other seven using 1 ng worth of DNA,0.5 uL of 100x BSA, 5 uL of NEB EcoRI 10xBuffer, 0.5 uL of NEB EcoRI, 0.5 uL of NEB PstI, and enough water to round out the volume. These digestions were put in the thermocycler for 30 minutes at 37˚C </li> | <li>Samples 2,4, and 7 were too dilute to be of use, but ran 50 uL digestions of the other seven using 1 ng worth of DNA,0.5 uL of 100x BSA, 5 uL of NEB EcoRI 10xBuffer, 0.5 uL of NEB EcoRI, 0.5 uL of NEB PstI, and enough water to round out the volume. These digestions were put in the thermocycler for 30 minutes at 37˚C </li> | ||
| + | <li> Ran 10 uL of each digestion reaction with 2 uL loading buffer versus a 1 kb ladder in a 0.8% agarose gel stained with EtBr. The resulting image is shown below, with columns containing the ladder, sample 2, sample 3, sample 5, sample 6, sample 8, sample 9, and sample 10 from left to right. </li> | ||
| + | <li> Samples 3 and 10 match the fragment sizes expected for R0011, while 5 might be pSB74, though the fragment size seems rather large. The other assignments are rather ambiguous, so gave up on identifying tubes. </li> | ||
<li> Redid inoculation of liquid cultures of strains containing pSB74, R0011, J23114, C0012, and B0015 and left in ampicillin LB on the shaker overnight. </li> | <li> Redid inoculation of liquid cultures of strains containing pSB74, R0011, J23114, C0012, and B0015 and left in ampicillin LB on the shaker overnight. </li> | ||
</ul> | </ul> | ||
| + | <i> This day's activities are also recorded on pages 18 and 19 of the hard copy lab notebook. </i> | ||
<!------------- Monday -------------> | <!------------- Monday -------------> | ||
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<li> | <li> | ||
| - | Tuesday | + | Tuesday 6/22--Miniprep redos |
</li> | </li> | ||
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<!------------- Tuesday -------------> | <!------------- Tuesday -------------> | ||
| - | + | <ul> | |
| + | <li>Began miniprep of overnight cultures, but in initial cell pelleting phase the centrifuge spun the tops of the tubes, spilling them. </li> | ||
| + | <li> Started over with the inoculation of two 5 mL liquid ampicillin LB cultures each of pSB74, R0011, C0012,J23114, and B0015 containing strains. </li> | ||
| + | <li> When OD is approximately 0.6, took 0.5 mL of each solution and mixed with 0.5 mL of 50% filter-sterilized glycerol and set aside for glycerol stocks. </li> | ||
| + | <li> Let cultures continue to grow all day, then miniprepped via the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> microcentrifuge protocol, </a> eluting into 40 uL of water. </li> | ||
| + | </ul> | ||
| + | <i> This day's activities are also recorded on page 18 of the hard copy lab notebook. </i> | ||
<!------------- Tuesday -------------> | <!------------- Tuesday -------------> | ||
</div> | </div> | ||
<li> | <li> | ||
| - | Wednesday | + | Wednesday 6/23--Making TSI agar slants & transformation of P0312 |
</li> | </li> | ||
<a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a> | <a id="link" href="javascript:ReverseDisplay('wednesday')">See more/less</a> | ||
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<!------------- Wednesday -------------> | <!------------- Wednesday -------------> | ||
| - | + | <b>Nanodrop of 6/22 miniprep </b> <br/> | |
| + | Nanodropped the ten minipreps from the day before and got the following results: | ||
| + | <td>Sample</td> | ||
| + | <td>J23114a</td><td>J23114b</td><td>C0012a</td><td>C0012b</td><td>R0011a</td><td>R0011b</td><td>B0015a</td><td>B0015b</td><td>pSB74a</td><td>pSB74b</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>DNA Concentration (ng/ul)</td> | ||
| + | <td> 122.9</td><td> 91.3 </td><td>17.4</td><td>57.9/td><td>86.8</td><td>59.5</td><td>70.2</td><td>83.7</td><td>67.3</td><td>44.2</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | Also flash froze in liquid N<sub>2</sub> the glycerol DNA solutions from 6/22 and put them in the -80˚C freezer. <br/> | ||
| + | <b> Triple Sugar Iron (TSI) agar preparation</b> <br/> | ||
| + | So that can readily determine whether bacteria are producing hydrogen sulfide, created a batch of TSI agar slants according to the following <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/TSI_agar"> recipe </a>, with ampicillin as the added antibiotic and tubes of the medium allowed to solidify on an angle to produce a characteristic slant. If a culture containing thiosulfate reductase is grown in such a slant, it will turn the medium black by precipitating out black iron sulfide, an obvious visual cue of hydrogen sulfide production. <br/> | ||
| + | <b> Transformation of BBa_P0312</b> <br/> | ||
| + | After realizing that Biobrick P0312 can be used in place of C0012 and R0011, so transformed BL21 with P0312 according to the <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation"> standard transformation protocol </a> | ||
| + | <i> This day's labwork is also recorded on pages 18, 20, and 21 of the hard copy lab notebook. </i> | ||
<!------------- Wednesday -------------> | <!------------- Wednesday -------------> | ||
</div> | </div> | ||
Revision as of 08:22, 27 October 2010
our project
lab notebook: week 3 (6/21-6/27)
- Monday 6/21--Miniprep Biobrick plasmids from overnight culture, but a mix up samples, necessitating a diagnostic digest. See more/less
- Tuesday 6/22--Miniprep redos See more/less
- Wednesday 6/23--Making TSI agar slants & transformation of P0312 See more/less
- Thursday See more/less
- Friday See more/less
- 6/21-AM miniprep but mixed up tubes--performed EcoR I/Pst I digest & gel to help distinguish tubes, though not all of them can be identified. Inoculated liquid cultures of transformants (again)
- 6/22 miniprep again
