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Team:UNIPV-Pavia/Notebook/Week7
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| - | |[[Image:pv_pcr_01_08.jpg|thumb| | + | |[[Image:pv_pcr_01_08.jpg|thumb|470px|left|Colony PCR for '''BBa_J23100'''-BBa_E0240, '''BBa_B0030'''-BBa_E0061: Marker and 5 colonies for each ligation]] |
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Revision as of 09:59, 20 July 2008
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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 7: 06/30/08 - 06/5/08
06/30/08
- We received sequencing results for BBa_B0030-BBa_C0061 (4th colony and 7th colony): sequences were correct! We decided to keep the 4th colony.
- Colony PCR for BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0061: 5 colonies for every plate. Gel showed many working colonies: we chose first colonies for the two ligations.
 |
- We infected 9 ml LB + Amp with 30 µl of:
| BBa_C0012 | BBa_B1006 | BBa_R0051-BBa_B0030 | BBa_B0030-BBa_C0061 |
| BBa_C0062 | BBa_J23100-BBa_E0240 (1) | BBa_B0030-BBa_C0078 (1) | BBa_J23100-BBa_B0030 |
| BBa_C0040 | BBa_I15010 |
- Tomorrow we will be ready to perform 5 ligations!
07/1/08
- Glycerol stocks for:
| BBa_C0012 | BBa_B1006 | BBa_R0051-BBa_B0030 | BBa_B0030-BBa_C0061 |
| BBa_C0062 | BBa_J23100-BBa_E0240 (1) | BBa_B0030-BBa_C0078 (1) | BBa_J23100-BBa_B0030 |
| BBa_C0040 | BBa_I15010 |
- Miniprep for all these parts.
- We sent BBa_J23100-BBa_E0240 (1) and BBa_B0030-BBa_C0078 (1) to Primm for sequencing.
- We performed digestion for:
| BBa_C0012 (X-P) | BBa_B1006 (E-X) | BBa_R0051-BBa_B0030 (S-P) | BBa_B0030-BBa_C0061 (E-S) |
| BBa_C0062 (X-P) | BBa_C0040 (X-P) | BBa_I15010 (X-P) | BBa_J23100-BBa_B0030 (S-P) |
- Gel run/cut for:
| BBa_C0012 (X-P) | BBa_C0062 (X-P) | BBa_B0030-BBa_C0061 (E-S) |
| BBa_C0040 (X-P) | BBa_I15010 (X-P) |
- Gel extraction for these 5 parts.
- DNA precipitation with sodium acetate for:
| BBa_B1006 (E-X) | BBa_R0051-BBa_B0030 (S-P) | BBa_J23100-BBa_B0030 (S-P) |
- Ligation:
- BBa_J23100-BBa_B0030-BBa_C0012
- BBa_J23100-BBa_B0030-BBa_C0040
- BBa_J23100-BBa_B0030-BBa_I15010
- BBa_R0051-BBa_B0030-BBa_C0062
- BBa_B0030-BBa_C0061-BBa_B1006
- We incubated ligation reaction at 16°C overnight.
07/2/08
- We transformed ligations (5 µl) and plated transformed bacteria.
- We also transformed BBa_B1006(E-X), BBa_J23100-BBa_B0030(S-P) and BBa_R0051-BBa_B0030 (1 µl) and plated transformed bacteria to estimate background noise.
- We received sequencing results for:
- BBa_B0030-BBa_E0040
- BBa_B0030-BBa_C0051
- BBa_B0030-BBa_E1010
- All the sequences were correct!
- We infected 9 ml LB + Amp with 30 µl of:
| BBa_B0030 | BBa_B0030-BBa_E0040 | BBa_B0030-BBa_C0051 |
| BBa_B1006 | BBa_B0030-BBa_E1010 |
- glycerol stocks.
07/3/08
- All the ligation plates showed carpets, while negative control plates showed a weak background noise.
- Single colonies plates for ligation plates.
- Glycerol stocks for:
| BBa_B0030 | BBa_B0030-BBa_E0040 | BBa_B0030-BBa_C0051 |
| BBa_B1006 | BBa_B0030-BBa_E1010 |
- Miniprep for these parts.
- We performed digestion:
| BBa_B0030 (E-X) | BBa_B0030-BBa_E0040 (E-S) | BBa_B0030-BBa_C0051 (E-S) |
| BBa_B1006 (E-X) | BBa_B0030-BBa_E1010 (E-S) |
- Gel run/cut for:
| BBa_B0030-BBa_E0040 (E-S) | BBa_B0030-BBa_C0051 (E-S) | BBa_B0030-BBa_E1010 (E-S) |
- Gel extraction.
- DNA precipitation with sodium acetate for:
| BBa_B0030 (E-X) | BBa_B1006 (E-X) |
Ligation:
- BBa_B0030-BBa_E0040-BBa_B1006
- BBa_B0030-BBa_C0051-BBa_B0030
- BBa_B0030-BBa_E1010-BBa_B1006
- We incubated ligation reactions at 16°C overnight.
07/4/08
- We transformed the 3 ligations (2 µl) and plated transformed bacteria.
- Colony PCR for single colonies plates (3 colonies for each plate):
| BBa_J23100-BBa_B0030-BBa_C0012 | BBa_J23100-BBa_B0030-BBa_C0040 | BBa_J23100-BBa_B0030-BBa_I15010 |
| BBa_R0051-BBa_B0030-BBa_C0062 | BBa_B0030-BBa_C0061-BBa_B1006 |
- Electrophoresis for PCR result: unfortunately, BBa_J23100-BBa_B0030-BBa_C0012 and BBa_J23100-BBa_B0030-BBa_I15010 did not show any true positive colony. We decided to re-perform colony PCR for these two parts next week. We chose to keep the first colonies for the other 3 ligations to grow 9 ml cultures overnight.
 |
07/5/08
- All the ligation plates showed colonies! Next week we will perform colony PCR to find true positive colonies.
- Glycerol stocks and Miniprep for: BBa_J23100-BBa_B0030-BBa_C0040 (1), BBa_R0051-BBa_B0030-BBa_C0062 (1), BBa_B0030-BBa_C0061-BBa_B1006 (1).
