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Team:UNIPV-Pavia/Notebook/Week4
From 2008.igem.org
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|BBa_J23100-'''BBa_E0240''' | |BBa_J23100-'''BBa_E0240''' | ||
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Revision as of 11:35, 6 July 2008
 Home
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|  The Project
|  Biological Safety
|  Parts Submitted to the Registry
|
|---|---|---|---|---|
 Dry Lab
|  Wet Lab
|  Modeling
|  Protocols
|  Activity Notebook
|
Notebook
| Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 |
|---|
Week 4: 06/09/08 - 06/13/08
06/09/08
- We prepared 4 glycerol stocks taking 800 µl from 9 ml cultures containing:
| BBa_B0030 (one of the two cultures) | BBa_R0051 | BBa_E0240 |
| BBa_J23100-BBa_B0030 |
|
|
- We performed digestion protocol to open plasmids:
| BBa_B0030 (1st culture) (E-X) | BBa_R0051 (S-P) | BBa_E0240 (E-X) |
| BBa_B0030 (2nd culture) (S-P) |
- We ran a gel with these parts.
- We performed gel extraction.
- Antarctic Phosphatase for these 4 parts.
- Ligation (30 µl final volume):
- BBa_J23100-BBa_E0240 (Pcon(E-S)-assGFP(E-X))
- BBa_R0051-BBa_B0030 (Plam(S-P)-RBS(X-P))
- BBa_B0030-BBa_E0040 (RBS(S-P)-GFP(X-P))
- BBa_B0030-BBa_C0051 (RBS(S-P)-cI(X-P))
- BBa_B0030-BBa_E1010 (RBS(S-P)-RFP(X-P))
- BBa_B0030-BBa_C0061 (RBS(S-P)-luxI_LVA(X-P))
- BBa_B0030-BBa_C0078 (RBS(S-P)-lasI(X-P))
- We incubated ligation overnight at 16°C.
06/10/08
- We plated the 7 ligations.
06/11/08
- BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 plates showed respectively 4 and 1 colonies, while the other plates didn't show any colony.
- We picked up one colony from the two working plates to grow 9 ml cultures of transformed bacteria overnight.
- We also infected 9 ml of LB + suitable antibiotic with 30 µl of:
| BBa_E0240 | BBa_B0030 | BBa_E0040 | BBa_E1010 |
| BBa_C0051 | BBa_C0061 | BBa_C0078 |
- glycerol stocks. We incubated all the 9 ml cultures overnight at 37°C.
- We contacted Francesca Ceroni from Bologna team for suggestions about ligation reaction. We decided to reduce reaction volume from 30 to 20 µl, increment insert:vector molar ratio from 1:2 to 1:3.
06/12/08
- We prepared 9 glycerol stocks taking 800 µl from 9 ml cultures containing:
| BBa_E0240 | BBa_B0030 | BBa_E0040 | BBa_R0051-BBa_B0030 |
| BBa_C0051 | BBa_C0061 | BBa_C0078 | BBa_J23100-BBa_E0240 |
| BBa_E1010 |
- We tested our microscope and BBa_J23100-BBa_E0240 fluorescence:
- We infected 150 µl of LB + Amp with 30 µl of BBa_J23100 glycerol stock (positive control)
- We took 30 µl of BBa_J23100-BBa_E0240 9 ml culture and infected 150 µl of LB + Amp.
- We incubated these two cultures at 37°C, 220 rpm for 1 hour and 30 min.
- Then, we took the 180 µl cultures and tried to watch them respectively through red and green fluorescence channel. BBa_J23100-BBa_E0240 didn't glow, while our positive control, BBa_J23100, glowed correctly through red channel.
 |
- Miniprep for these 9 parts.
- PCR for BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 to check for contaminations (even this time, we could not check the actual insert length because insert in ligation reaction were too small).
- We performed electrophoresis on PCR result to check for contaminating plasmids: the plasmids was correct.
- We sent purified BBa_J23100-BBa_E0240 and BBa_R0051-BBa_B0030 to Primm for sequencing.
