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Team:UNIPV-Pavia/Notebook/Week10
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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 10: 07/21/08 - 07/25/08
07/21/08
- Colony PCR for BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079: 6 colonies from single colonies plate.
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- Gel results: all the 6 picked colonies were true positive! we chose the 5th colont to grow a 9 ml overnight culture.
- We also infected 9 ml LB + Amp with 30 µl of BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0061 glycerol stocks to grow two overnight cultures.
- Ligation:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1:2 ratio, 40 ng of vector)
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (30 ng of vector)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
- We incubated ligations at 16°C overnight.
07/22/08
- Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.
- Glycerol stocks/miniprep for:
| BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 | BBa_B0030-BBa_C0061 |
| BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 |
- We sent BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 purified plasmids to Primm for sequencing.
- Plasmid digestion for:
| BBa_B0030-BBa_C0061 (E-S) |
| BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 (E-X) |
- Gel run/cut/gel extraction.
07/23/08
- Ligation plates showed colonies!We put ligation plates at +4°C.
- Ligation for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079.
- We incubated ligation at 16°C overnight.
07/24/08
- Transformation (1 µl) for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
- Colony PCR for:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
- (We performed one PCR for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
- Electrophoresis (1.2% agarose) for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082:
 |
- Electrophoresis (1% agarose) for BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 and BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006:
 |
- Gel results:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 1st colony
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 1st colony
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 1st and 3rd colony
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 1st and 4th colony
- We decided to keep two colonies for BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 and BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
- We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.
07/25/08
- Glycerol stocks/miniprep for:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1)
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
- We sent these purified plasmids:
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)
- to Primm for sequencing.
