Team:Paris Saclay/Notebook/July/22

From 2014.igem.org

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=Tuesday 22st July=
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{{Team:Paris_Saclay/notebook_header}}
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=Tuesday 22nd July=
==Lab Work==
==Lab Work==
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===The chassis coli Odor free===
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====Preparation and transformation of competent cells MG1655 and MG1655Z1 by electroporation====
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''by Arnaud''
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 +
Dilute 300µl of bacterial culture in 30ml LB medium at 30°C.
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 +
Shake vigorously at 30°C until the OD600nm reaches 0,6.
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When the OD<sub>600</sub> reached 0.6, proceed to the [https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_competent_cells_by_electroporation electroporation].
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===1 - Samples for stock===
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====2 - Samples for stock====
''by Fabio''
''by Fabio''
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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]
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===2 - Electrophoresis===
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====3 - Electrophoresis====
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''by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)''
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''by Arnaud (Process A), Fabio (gel), Marie and Romain (Process A)''
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[[File:Electrophoresis_22.07.14.jpg|400px|right]]
We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.
We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.
'''''Process A'''''
'''''Process A'''''
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#
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#Empty
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#
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#p cola Ges
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#DY330 pJBEI I
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#
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#DY330 pJBEI II
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#
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#BT340
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#
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#
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#
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#
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#
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#
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#
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'''''Process B'''''
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#
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#
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'''Results:'''
'''Results:'''
*'''A''':
*'''A''':
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*'''B''':
 
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*'''C''':
 
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Protocol]
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===3 - Bacterial Culture===
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====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.
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===4 - Plasmid DNA extraction===
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====5 - Plasmid DNA extraction====
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''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>
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==<span style="color:blue">C - Lemon scent</span>==
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===B - Lemon scent===
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====Extraction of p cola plasmid DNA====
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''by Sean''
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===PCR Targeting===
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p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids.
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====PCR Targeting====
''by Romain''
''by Romain''
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Strains used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)
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Strains used: DY330 (clone I and II). Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)
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'''Protocol:''' (For each clone)
Step 1 - Dilution of 300µl of bacterial culture in 30ml of LB + 30µl Cm at 30°C.
Step 1 - Dilution of 300µl of bacterial culture in 30ml of LB + 30µl Cm at 30°C.
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Step 3 - When the culture OD<sub>650</sub> = 0,6 (Step 1):
Step 3 - When the culture OD<sub>650</sub> = 0,6 (Step 1):
*put in ice during 10min.
*put in ice during 10min.
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*centriguge 4°C, 5min, 4000rpm
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*centriguge 4°C, 5min, 4000rpm.
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*Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% '''COLD'''
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*Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% '''COLD'''.
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*centriguge 4°C, 5min, 4000rpm.
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*Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''.
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*centriguge 4°C, 5min, 4000rpm.
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*Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% '''COLD'''.
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Step 4 - Transformation:
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*control 50µl (without DNA)
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*50µl transformed bacteria + 2,5µl plasmid + 7,5µl pOSV 230 PCR (purifié)
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Step 5 - Electroporation control
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Step 6 - Spread on ApraR and CmR dishes
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*Control dishes : 100µl ND
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*1ml of bacterial culture (250µl on each dish)
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Results: [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24 24th July]
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==Reunion==
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Conference with the team to discuss about the project. The handled topics were:
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* Decisions about our new Visual Identity
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* Order of the t-shirts and sweaters for the team
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* Discussion about the main message of the project
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* Presentation's planning of the project
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* Exhibition of our draft work
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==Photo of the Day==
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[[File:Paris Saclay 22_july.jpg|600px|center]]
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'''Members there''':
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'''People there''':
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* Instructors and advisors: Solenne.
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* Instructors and advisors: Solenne and Sylvie.
* Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.
* Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.
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[https://2014.igem.org/Team:Paris_Saclay/Notebook Back to the calendar]
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{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:25, 14 October 2014

Contents

Tuesday 22nd July

Lab Work

The chassis coli Odor free

Preparation and transformation of competent cells MG1655 and MG1655Z1 by electroporation

by Arnaud

Dilute 300µl of bacterial culture in 30ml LB medium at 30°C.

Shake vigorously at 30°C until the OD600nm reaches 0,6.

When the OD600 reached 0.6, proceed to the electroporation.

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 A), Fabio (gel), Marie and Romain (Process A)

Electrophoresis 22.07.14.jpg

We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.

Process A

  1. Empty
  2. p cola Ges
  3. DY330 pJBEI I
  4. DY330 pJBEI II
  5. BT340

Results:

  • A:

Protocol

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.

B - Lemon scent

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.

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

Photo of the Day

600px

People there:

  • Instructors and advisors: Solenne and Sylvie.
  • Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/July/22"