Team:UNIPV-Pavia/Notebook/Week6
From 2008.igem.org
(Difference between revisions)
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**'''BBa_J23100'''-BBa_E0240 | **'''BBa_J23100'''-BBa_E0240 | ||
**'''BBa_B0030'''-BBa_C0078 | **'''BBa_B0030'''-BBa_C0078 | ||
+ | |||
+ | <br><br> | ||
+ | '''06/25/08''' | ||
+ | <br> | ||
+ | *We transformed 5 µl of the two ligations. | ||
+ | |||
+ | *We also transformed 0.5 µl of BBa_B0030(S-P) and BBa_J23100(S-P) plasmids to estimate background noise. | ||
+ | |||
+ | *Colony PCR (6 colonies for each plate) for: | ||
+ | **'''BBa_B0030'''-BBa_E0040 | ||
+ | **'''BBa_B0030'''-BBa_E1010 | ||
+ | **'''BBa_B0030'''-BBa_C0051 | ||
+ | |||
+ | {| | ||
+ | |[[Image:pv_pcr_04_05_06.jpg|thumb|300px|left|Colony PCR for '''BBa_B0030'''-BBa_E0040, '''BBa_B0030'''-BBa_E0051 and '''BBa_B0030'''-BBa_E1010: Marker and 6 colonies for each ligation]] | ||
+ | |} |
Revision as of 22:53, 6 July 2008
Home | The Team | The Project | Biological Safety | Parts Submitted to the Registry |
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Dry Lab | Wet Lab | Modeling | Protocols | Activity Notebook |
Notebook
Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 |
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Week 5: 06/23/08 - 06/20/08
06/23/08
- Colony PCR for BBa_B0030-BBa_C0061 and BBa_B0030 (no Ant.Phosph.)-BBa_C0061: 10 colonies for every plate.
- We ran PCR results: some colonies showed both ligated plasmid (heavier band) and false positive plasmid, because it was difficult to pick up single colonies from bacteria carpet. Ligation with Antarctic Phosphatase showed non-pure colonies and 3 false positives. Ligation without Antarctic Phosphatase showed 3 non-pure colonies and 7 pure colonies.
- We decided to avoid Antarctic Phosphatase treatment in next ligations to save time.
- We also decided to cut up to 1 µg of vector, to reduce background noise.
- We choose 4th and 7th colonies to grow 9 ml cultures overnight.
- We also infected 9 ml LB + Amp with 30 µl of:
BBa_E0240 | BBa_C0078 | BBa_B0030 | BBa_J23100 |
- glycerol stocks. We incubated all the 6 cultures at 37°C, 220 rpm.
- We transformed 10 µl of the remaining ligations:
- BBa_B0030-BBa_E0040
- BBa_B0030-BBa_E1010
- BBa_B0030-BBa_C0051
- Wiki updating: Project section.
06/24/08
- All the 3 ligation plates showed carpets. We decided to streak plates to produce single colonies plates. We incubated the 3 single colonies plates at 37°C overnight.
- Glycerol stocks for:
BBa_E0240 | BBa_C0078 | BBa_B0030-BBa_C0061(4) |
BBa_J23100 | BBa_B0030 | BBa_B0030-BBa_C0061(7) |
- Miniprep for these 6 plasmids.
- Plasmid digestion:
BBa_E0240 (X-P) | BBa_C0078 (X-P) |
BBa_J23100 (S-P) | BBa_B0030 (S-P) |
- DNA precipitation with sodium acetate for BBa_J23100 (S-P) and BBa_B0030 (S-P).
- Gel run for BBa_E0240 (X-P) and BBa_C0078 (X-P)
- Gel extraction.
- Ligation:
- BBa_J23100-BBa_E0240
- BBa_B0030-BBa_C0078
06/25/08
- We transformed 5 µl of the two ligations.
- We also transformed 0.5 µl of BBa_B0030(S-P) and BBa_J23100(S-P) plasmids to estimate background noise.
- Colony PCR (6 colonies for each plate) for:
- BBa_B0030-BBa_E0040
- BBa_B0030-BBa_E1010
- BBa_B0030-BBa_C0051