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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 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
Week 10: 07/21/08 - 07/25/08
07/21/08
- Colony PCR for B0030-C0078-B1006-R0079: 6 colonies from single colonies plate.
 |
- Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.
- We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-R0040 and B0030-C0061 glycerol stocks to grow two overnight cultures.
- Ligation:
- J23100-B0030-C0012-B1006 (1:2 ratio, 40 ng of vector)
- B0030-I15009-B1006-R0082 (30 ng of vector)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-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:
| J23100-B0030-C0040-B1006-R0040 | B0030-C0061 |
| B0030-C0078-B1006-R0079 |
- We sent J23100-B0030-C0040-B1006-R0040 and B0030-C0078-B1006-R0079 purified plasmids to Primm for sequencing.
- Plasmid digestion for:
| B0030-C0061 (E-S) | B0030-C0078-B1006-R0079 (E-X) |
- Gel run/cut/gel extraction.
07/23/08
- Ligation plates showed colonies!We put ligation plates at +4°C.
- Ligation for B0030-C0061-B0030-C0078-B1006-R0079.
- We incubated ligation at 16°C overnight.
07/24/08
- Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
- Colony PCR for:
- J23100-B0030-C0012-B1006
- B0030-I15009-B1006-R0082
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
- (We performed one PCR for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
 |  |
- Electrophoresis (1.2% agarose) for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082:
 |
- Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006:
 |
- Gel results:
- J23100-B0030-C0012-B1006 1st colony
- B0030-I15009-B1006-R0082 1st colony
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 1st and 4th colony
- We decided to keep two colonies for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-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:
- J23100-B0030-C0012-B1006 (1)
- B0030-I15009-B1006-R0082 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
- We sent these purified plasmids:
- B0030-I15009-B1006-R0082 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
- to Primm for sequencing.
- We put B0030-C0061-B0030-C0078-B1006-R0079 plate at +4°C.
