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Team:Paris Saclay/Notebook/July/22
From 2014.igem.org
(→C - Lemon scent) |
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==Lab Work== | ==Lab Work== | ||
| - | ===1 - Extraction of p cola plasmid DNA=== | + | ====1 - Extraction of p cola plasmid DNA==== |
''by Sean'' | ''by Sean'' | ||
p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids. | p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids. | ||
| - | ===2 - Samples for stock=== | + | ====2 - Samples for stock==== |
''by Fabio'' | ''by Fabio'' | ||
| Line 27: | Line 27: | ||
from Liquide Bacterial Cultures transformed the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/21 21st July] | from Liquide Bacterial Cultures transformed the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/21 21st July] | ||
| - | ===3 - Electrophoresis=== | + | ====3 - Electrophoresis==== |
''by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)'' | ''by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)'' | ||
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[https://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Protocol] | [https://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Protocol] | ||
| - | ===4 - Bacterial Culture=== | + | ====4 - Bacterial Culture==== |
''by Fabio and Marie'' | ''by Fabio and Marie'' | ||
One liquid culture with 5ml of LB (37°C - 150 rpm), IL1403 from the concentrated colony. | One liquid culture with 5ml of LB (37°C - 150 rpm), IL1403 from the concentrated colony. | ||
| - | ===5 - Plasmid DNA extraction=== | + | ====5 - Plasmid DNA extraction==== |
''by Sean and Terry'' | ''by Sean and Terry'' | ||
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<span style="color:red">Note: we accidentally used a buffer without 10% ethanol. After removal of the buffer, we repeated the step with a correct version. This may have affected the electrophoresis which followed.</span> | <span style="color:red">Note: we accidentally used a buffer without 10% ethanol. After removal of the buffer, we repeated the step with a correct version. This may have affected the electrophoresis which followed.</span> | ||
| - | == | + | ===C - Lemon scent=== |
| - | ===PCR Targeting=== | + | ====PCR Targeting==== |
''by Romain'' | ''by Romain'' | ||
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Results: [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24 24th July] | Results: [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24 24th July] | ||
| + | |||
| + | ==Reunion== | ||
| + | Conference with the team to discuss about the project. The handled topics were: | ||
| + | * Decisions about our new Visual Identity | ||
| + | * Order of the t-shirts and sweaters for the team | ||
| + | * Discussion about the main message of the project | ||
| + | * Presentation's planning of the project | ||
| + | * Exhibition of our draft work | ||
'''Members there''': | '''Members there''': | ||
Revision as of 12:21, 24 July 2014
Contents |
Tuesday 22nd July
Lab Work
1 - Extraction of p cola plasmid DNA
by Sean
p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids.
2 - Samples for stock
by Fabio
We collected 1ml of each sample, added 240µl of glycerol (87%) and stored at -80°C.
- BBa_K1033902
- BBa_K1033905
- BBa_K1033910
- BBa_K1033913
- BBa_K1033922
- BBa_K1033925
- BBa_K1033927
- BBa_K1033929
- BBa_K103921
- BBa_J45017
- BBa_K731201
- BBa_K762100
from Liquide Bacterial Cultures transformed the 21st July
3 - Electrophoresis
by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)
We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.
Process A
Process B
Results:
- A:
- B:
- C:
4 - Bacterial Culture
by Fabio and Marie
One liquid culture with 5ml of LB (37°C - 150 rpm), IL1403 from the concentrated colony.
5 - Plasmid DNA extraction
by Sean and Terry
Plasmids used: cf part 1
Note: we accidentally used a buffer without 10% ethanol. After removal of the buffer, we repeated the step with a correct version. This may have affected the electrophoresis which followed.
C - Lemon scent
PCR Targeting
by Romain
Strains used: DY330 (clone I and II). Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)
Protocol: (For each clone)
Step 1 - Dilution of 300µl of bacterial culture in 30ml of LB + 30µl Cm at 30°C.
Step 2 - With the pre-culture:
- make glycerol stock: 1ml bacterial culture with 240µl glycerol 87%.
- Make extraction of 5ml of plasmid Protocol
Step 3 - When the culture OD650 = 0,6 (Step 1):
- put in ice during 10min.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% COLD.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.
Step 4 - Transformation:
- control 50µl (without DNA)
- 50µl transformed bacteria + 2,5µl plasmid + 7,5µl pOSV 230 PCR (purifié)
Step 5 - Electroporation control
Step 6 - Spread on ApraR and CmR dishes
- Control dishes : 100µl ND
- 1ml of bacterial culture (250µl on each dish)
Results: 24th July
Reunion
Conference with the team to discuss about the project. The handled topics were:
- Decisions about our new Visual Identity
- Order of the t-shirts and sweaters for the team
- Discussion about the main message of the project
- Presentation's planning of the project
- Exhibition of our draft work
Members there:
- Instructors and advisors: Solenne.
- Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.
