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Team:UNIPV-Pavia/Notebook/Week5
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Notebook
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Week 5: 06/16/08 - 06/20/08
06/16/08
- We picked up the only colony in BBa_B0030-BBa_C0078 plate to grow a 9 ml culture of transformed bacteria overnight.
- We also infected 9 ml of LB + suitable antibiotic with 30 µl of:
| BBa_B0030 | BBa_E1010 | BBa_E0040 |
| BBa_C0061 | BBa_C0051 |
- glycerol stocks. We incubated the 9 ml culture overnight at 37°C, 220 rpm.
06/17/08
- Glycerol stocks for:
| BBa_B0030 | BBa_E1010 | BBa_E0040 |
| BBa_C0061 | BBa_C0051 | BBa_B0030-BBa_C0078 |
- Miniprep for all these parts.
- Digestion for:
| BBa_B0030 (S-P) | BBa_E1010 (X-P) | BBa_E0040 (X-P) |
| BBa_C0061 (X-P) | BBa_C0051 (X-P) | BBa_B0030-BBa_C0078 (X-S) |
- Gel run for BBa_B0030-BBa_C0078 to check for insert length: unfortunately, there was not a band where we expected...the only colony was a false positive. We'll try to ligate it in the next days.
- Gel run for:
| BBa_B0030 (S-P) | BBa_E1010 (X-P) | BBa_E0040 (X-P) |
| BBa_C0061 (X-P) | BBa_C0051 (X-P) |
- Gel cut and DNA extraction.
- We put DNA at -20°C. The next day we will perform some ligation reaction in different conditions, looking for the best protocol.
06/18/08
- We planned the following ligation experiments:
- Transformation with BBa_B0030(S-P), to check background noise (we will know the amount of not digested vector);
- Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and no insert;
- Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and no insert;
- Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and BBa_C0061 insert;
- Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0061 insert;
- 3 Transformation with these ligations: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0051, BBa_E1010, BBa_E0040 inserts;
- Antarctic Phosphatase for half of BBa_B0030 (S-P) volume.
- We transformed 60 µl of TOP10 with 1 µl of BBa_B0030 (S-P)
- We plated transformed bacteria and incubated them at 37°C overnight.
- Antarctic Phosphatase for half a BBa_B0030 (S-P) volume.
- Ligation (10 µl final volume):
- BBa_B0030 alone
- BBa_B0030 (no Ant.Phosph.) alone
- BBa_B0030(no Ant.Phosph.)-BBa_C0061
- BBa_B0030-BBa_C0061
- BBa_B0030-BBa_C0078
- BBa_B0030-BBa_E0040
- BBa_B0030-BBa_E1010
- We gave a lecture about Synthetic Biology and our current work at DIS (Department of Informatics and System Science).
06/19/08
- We received sequencing results for
