<a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0030">BBa_B0030</a> (S-P)</td>
<td>1</td>
<td></html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_I13501</a> (X-P)</td>
<td>7</td>
<td>1</td>
<td>1</td>
</tr>
<tr>
<td>E6</td>
<td><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0031">BBa_B0031</a> (S-P)</td>
<td>1</td>
<td><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_I13501</a> (X-P)</td>
<td>7</td>
<td>1</td>
<td>1</td>
</tr>
<tr>
<td>E7</td>
<td><a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> (S-P)</td>
<td>1</td>
<td><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_I13501</a> (X-P)</td>
<td>7</td>
<td>1</td>
<td>1</td>
</tr>
</table>
</center>
<a name="July.2C_6th"></a> July, 6th
<a href="http://partsregistry.org/wiki/index.php/Part:BBa_C0261">BBa_C0261</a> was resuspended from iGEM 2011 kit distribution, Plate 1, well 14C in 15 μl of ddH2O; <a href="http://partsregistry.org/wiki/index.php/Part:BBa_C0261">BBa_C0261</a>, E1, E2, E3, E4, E5, E6, E7, and E8 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.
500 ml of LB without antibiotic were prepared.
<a href="#indice" title="">^top</a>
<a name="July.2C_7th"></a> July, 7th
All plates showed a lot of colonies, except for E-8 (5 colonies); two colonies for E1, E2, E3, E4, E5, E6, E7 plates and three colonies for E8 plate were picked and inoculated in 10 μl LB for inoculum and screening PCR.
A 20x mix was prepared for PCR reaction:
| H2O (μl) |
Buffer 10x (μl) |
MgCl2 (μl) |
VF2 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_G00100">BBa_G00100</a>) μl |
VR (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_G00101">BBa_G00101</a>) μl |
dNTPs (μl) |
Taq polymerase (μl) |
| 360 |
50 |
20 |
10 |
10 |
10 |
20 |
24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:
- 94°C 30 seconds (denaturing)
- 60°C 1 minute (annealing)
- 72°C 1 minute and 20 seconds (elongation)
35 cycles were performed.
Liquid cultures were then inoculated in 800 μl LB with the proper antibiotic.
A small size and a medium size agarose gel were prepared.
After the PCR, gel run was carried out on the samples:
</html> Medium size gel  Small size gel
Except for E2-1, E4-1 and E7-1, other band lengths were correct; 750 μl of E1-2, E2-2, E3-1, E4-2, E5-2, E6-1, E7-2 and E8-3 liquid cultures were used to prepare glycerol stocks while the remaining 50 μl were refilled to 5 ml for plasmid purification and incubated at 37°C 220 rpm. BBa_R0040, BBa_C0261 and BBa_I13521 were also inoculated.
In the late afternoon the team met to talk about the wet-lab activity, the abstract and the bio-safety section.
July, 8th
Cultures were grown till saturation; plasmid purification was carried out:
| Plasmid |
DNA (ng/μl) |
| E1-2 |
64.1 |
| E2-2 |
56.2 |
| E3-1 |
53.5 |
| E4-2 |
67.2 |
| E5-2 |
73.5 |
| E6-1 |
92.2 |
| E7-2 |
67.6 |
| E8-3 |
25.3 |
| BBa_R0040 |
48.1 |
| BBa_C0261 |
61.2 |
| BBa_I13521 |
79.3 |
| 
"
