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Team:UNIPV-Pavia/Notebook/Week5
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Notebook
| 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 17 | Week 18 | Week 19 | Week 20 | Week 21 |
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:
- BBa_J23100-BBa_E0240 (4 samples from 4 different colonies): all the 4 colonies were false positives
- BBa_J23100-BBa_B0030: the sequence was correct!
- BBa_R0051-BBa_B0030: the sequence was correct!
- BBa_B0030(S-P) plate showed many colonies. We expect to find at least this amount of colonies in ligation plates.
- We heated ligation at 65°C for 10 min to inactivate T4 Ligase.
- We transformed 10 µl of the following ligations:
- BBa_B0030 alone
- BBa_B0030(no Ant.Phosph.) alone
- BBa_B0030 (no Ant.Phosph.)-BBa_C0061
- BBa_B0030-BBa_C0061
- We didn't transform the other 3 ligations because we wanted to check plated transformation the next day, to save 3 agar plates if the experiment doesn't work.
06/20/08
- Transformation results:
- BBa_B0030 alone showed many colonies (less than BBa_B0030(S-P) seen the previous day)
- BBa_B0030(no Ant.Phosph.) alone showed many colonies (the same quantity of BBa_B0030(S-P) seen the previous day)
- BBa_B0030 (no Ant.Phosph.)-BBa_C0061 showed a carpet
- BBa_B0030-BBa_C0061 showed a carpet
- Now we are happy with these plates! Next week we will check insert length by colony PCR/electrophoresis.
