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Team:UCL/Labbook/Week14
From 2013.igem.org
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</p> | </p> | ||
| + | <p class="body_text"> | ||
| + | Ligation 2 | ||
| + | </p> | ||
| + | |||
| + | <table> | ||
| + | <tr> | ||
| + | <th>Contents</th> | ||
| + | <th>ng/ul</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>pSB1C3 digested & pruified</td> | ||
| + | <td>25</td> | ||
| + | </tr> | ||
| + | <td>seo digested & purified</td> | ||
| + | <td>77</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>cmv digested & purified</td> | ||
| + | <td>25</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </p> | ||
| + | |||
| + | <div class="gap"> | ||
</div> | </div> | ||
| + | <p class="body_text"> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th>CMV (25 ng/ul)</th> | ||
| + | <th>1 c (3:1 molar ratio)</th> | ||
| + | <th>2 c (2:1 molar ratio)</th> | ||
| + | <th>3 c (1:1 molar ratio)</th> | ||
| + | <th>4 c (3:1 molar ratio)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Water (ul)</td> | ||
| + | <td>1.8</td> | ||
| + | <td>3.2</td> | ||
| + | <td>8</td> | ||
| + | <td>6</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Quick ligase buffer (ul)</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Backbone (ul)</td> | ||
| + | <td>4</td> | ||
| + | <td>4</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>*Insert (ul)</td> | ||
| + | <td>4.2</td> | ||
| + | <td>2.8</td> | ||
| + | <td>1</td> | ||
| + | <td>3</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Ligase (ul)</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>water (ul)</td> | ||
| + | <td>1.8</td> | ||
| + | <td>3.2</td> | ||
| + | <td>8</td> | ||
| + | <td>6</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Colony count</td> | ||
| + | <td>50</td> | ||
| + | <td>100+</td> | ||
| + | <td>100+</td> | ||
| + | <td>150+</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </p> | ||
| + | <p class="body_text"> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th>Zeocin (77 ng/ul)</th> | ||
| + | <th>5 z (3:1 molar ratio)</th> | ||
| + | <th>6 z (2:1 molar ratio)</th> | ||
| + | <th>7 z (1:1 molar ratio)</th> | ||
| + | <th>8 z (3:1 molar ratio)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Water (ul)</td> | ||
| + | <td>2.1</td> | ||
| + | <td>3.3</td> | ||
| + | <td>8</td> | ||
| + | <td>8</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Quick ligase buffer (ul)</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | <td>10</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Backbone (ul)</td> | ||
| + | <td>4</td> | ||
| + | <td>4</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>*Insert (ul)</td> | ||
| + | <td>3.9 from *</td> | ||
| + | <td>2.6 from *</td> | ||
| + | <td>1 from *</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Ligase (ul)</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | <td>21</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>water (ul)</td> | ||
| + | <td>1.8</td> | ||
| + | <td>3.2</td> | ||
| + | <td>8</td> | ||
| + | <td>6</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Colony count</td> | ||
| + | <td>30</td> | ||
| + | <td>0</td> | ||
| + | <td>1</td> | ||
| + | <td>0+</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </p> | ||
| + | |||
| + | </div> | ||
<!-- END CONTENT ------------------------------------------------------------------------------------------------------> | <!-- END CONTENT ------------------------------------------------------------------------------------------------------> | ||
Revision as of 20:16, 3 October 2013
Lab Weeks
Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16
Week 14
Monday 2nd September
We re-autoclaved water due to our suspicion it was nuclease contaminated. New inoculations (in 3 ml LB) of the transformations were prepared: 5 using cmv ligation and 5 using the zeocin ligation. These were left over night into the incushaker.
Tuesday 3rd September
The falcons were removed from the incu-shaker. Glycerol stocks were prepared using 0.5 ml while the rest was kept for minipreping.
Preparative digest of zeocin and K812014 plasmid
| Components (ul) | Prep digest of Zeocin | Prep digest of K812014 |
|---|---|---|
| Zeo/K812014 | 80+ | 20+ |
| EcoRI | 10 | 5 |
| PstI | 10 | 5 |
| Buffer 3 | 15 | 5 |
| BSA | 3 | 1 |
| dH2O | 22 | 14 |
| Total | 150 | 50 |
These were incubated for 1 hour at 37º C and for 20 min heat inactivated.
Nanodrop of the minipreps from the new inoculations.
| pDNA new | ng/ul | 260/280 |
|---|---|---|
| 51/1 | 9.5 | 2.2 |
| 51/2 | 9.9 | 1.91 |
| 51/3 | 8.9 | 1.93 |
| 51/4 | 8.6 | 2.31 |
| 51/5 | 13.2 | 2.49 |
| 45/1 | 8.2 | 1.89 |
| 45/2 | 8.7 | 2.65 |
| 45/3 | 10.7 | 2.13 |
| 45/4 | 9.1 | 2.00 |
| 45/5 | 9.7 | 2.34 |
| 28 glycerol stock | 40.4 | 2.23 |
| 45 glycerol stock | 36.7 | 2.24 |
| 51 glycerol stock | 45.4 | 2.18 |
| 52 glycerol stock | 28.4 | 1.98 |
These samples of minipreps and from glycerol stocks were run on a gel in order to check if the circularised potentially recombinant plasmid is present. Again, there were no bands for any of these samples, hence we tend to believe the ligations were not successful OR/AND the transformation was faulty.
Gel extraction - of digested E and P - K812014 biobrick plasmid After gel extraction purification of the corresponding 2 kb band, the concentration showed up to around 6.6 ng/ul and 260/280 = 1.73. Given the very small concentration, a second attempt of obtaining pure pSB1C3 was performed, not successful though, only 4.9 ng/ul concentration.
- of PCR purified zeocin, 2 kb band. This was purified and the nanodrop showed a concentation of 77.6 ng/ul and 260/280 = 1.82.
Nanodrop of E, P digested cmv (from 30 of August) gave a concentration of 25.1 ng/ul, 260/280 = 1.86.
New inoculations of K812014 biobrick were prepared and left for incubation over night in the incu-shaker (37 degrees Celsius, at 200 rpm).
Wednesday 4th September
Miniprep of the K812014 biobrick inoculations showed the following concentrations:
| Tube number | ng/ul | 260/280 |
|---|---|---|
| 1 | 151 | 1.93 |
| 2 | 80.8 | 1.93 |
| 3 | 111.5 | 1.82 |
| 4 | 219.1 | 1.94 |
Samples 1 and 2 went through a preparative digest
| item | ul |
|---|---|
| pDNA | 50 |
| Buffer 3 | 10 |
| EcoR1 | 7 |
| Pst1 | 7 |
| BSA | 2 |
| Water | 24 |
| Total | 100 |
Ligation 2
| Contents | ng/ul |
|---|---|
| pSB1C3 digested & pruified | 25 | seo digested & purified | 77 |
| cmv digested & purified | 25 |
| CMV (25 ng/ul) | 1 c (3:1 molar ratio) | 2 c (2:1 molar ratio) | 3 c (1:1 molar ratio) | 4 c (3:1 molar ratio) |
|---|---|---|---|---|
| Water (ul) | 1.8 | 3.2 | 8 | 6 |
| Quick ligase buffer (ul) | 10 | 10 | 10 | 10 |
| Backbone (ul) | 4 | 4 | 1 | 1 |
| *Insert (ul) | 4.2 | 2.8 | 1 | 3 |
| Ligase (ul) | 1 | 1 | 1 | 1 |
| Total | 21 | 21 | 21 | 21 |
| water (ul) | 1.8 | 3.2 | 8 | 6 |
| Colony count | 50 | 100+ | 100+ | 150+ |
| Zeocin (77 ng/ul) | 5 z (3:1 molar ratio) | 6 z (2:1 molar ratio) | 7 z (1:1 molar ratio) | 8 z (3:1 molar ratio) |
|---|---|---|---|---|
| Water (ul) | 2.1 | 3.3 | 8 | 8 |
| Quick ligase buffer (ul) | 10 | 10 | 10 | 10 |
| Backbone (ul) | 4 | 4 | 1 | 1 |
| *Insert (ul) | 3.9 from * | 2.6 from * | 1 from * | 1 |
| Ligase (ul) | 1 | 1 | 1 | 1 |
| Total | 21 | 21 | 21 | 21 |
| water (ul) | 1.8 | 3.2 | 8 | 6 |
| Colony count | 30 | 0 | 1 | 0+ |
