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Team:Yale/Our Project/Notebook/Week 3
From 2010.igem.org
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| - | Monday | + | Monday 6/21--Miniprep Biobrick plasmids from overnight culture, but a mix up samples, necessitating a diagnostic digest. |
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| - | + | <ul> | |
| + | <li>Miniprepped the cultures grown overnight according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/miniprep"> standard microcentrifuge protocol, </a> but mixed up tubes so hope to sort them out with a diagnostic double digest</li> | ||
| + | <li> The similar size and identical backgrounds of the Biobricks makes many of them difficult to distinguish by restriction digest failing cut sites within the Biobricks themselves. EcoRI and PstI are available but B0015, C0012, and J23114 (all 35 bp in around 3 kb backgrounds) are too hard to tell apart by such a digestion. However, a EcoRI/PstI digest should be able to tell them from R0011 and pSB74--the former will be divided into 1.1 kb and 2.1 kb fragments while the latter will end up as 3.6 kb and 9.4 kb fragments.</li> | ||
| + | <li> Before setting up digestion, used nanodrop to determine the concentration of the ten unknown samples (two of each plasmid) </li> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td>Sample</td> | ||
| + | <td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>DNA Concentration (ng/ul)</td> | ||
| + | <td> 22.0 </td><td> 76.1 </td><td>51.5</td><td>15.5</td><td>98.7</td><td>303.0</td><td>27.9</td><td>11.4</td><td>82.6</td><td>306.6</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <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> 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> | ||
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Revision as of 05:54, 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 See more/less
- Wednesday 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
