Team:Paris Saclay/Notebook/July/3

From 2013.igem.org

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<u>Culture confirmation</u>
 
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<br>
 
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<p>We performed another colonies seeding. 2 colonies selected from each Petri dish were seeded in liquid medium with their corresponding antibiotic at 37°C under stirring during 1 night.</p>
 
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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : July 1'''=
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='''Notebook : July 3'''=
=='''Lab work'''==
=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Objective : obtaining Bba_K1155000'''====
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===='''Objective : obtaining BBa_K1155000'''====
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===='''1 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009'''====
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===='''1 - Colony PCR of BBa_K1155000 to check good insertion of Pndh* in pSB1C3'''====
Abdou, Sheng, Zhou
Abdou, Sheng, Zhou
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DE;" |
| style="border:1px solid black;padding:5px;background-color:#DE;" |
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Tranformation of 07/02/13 works. We will do a Colony PCR of colonies.
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Tranformation of 07/02/13 works. We will do a PCR colony.
|}
|}
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Colonies count :  
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Colonies count for BBa_K1155000 :  
* Standard concentration :  
* Standard concentration :  
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** Bba_K1155000 : 0
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** BBa_K1155000 : 0 colony
* High concentration :  
* High concentration :  
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** Bba_K1155000 : 2
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** BBa_K1155000 : 2 colonies
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[[File:Ps0307jour.jpg|300px]]
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<br>
<br>
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<center>[[File:PSprimer07.jpg|right|300px]]</center>
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<center>[[File:PSprimer07.jpg|right|250px]]</center>
<br>
<br>
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'''Primer and PCR :'''
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'''[https://2013.igem.org/Team:Paris_Saclay/Primers Primers] and PCR :'''
<p>'''VF2, VR, Pfnr_up, Pfnr_down are four oligos that we used for plasmid amplification. We used tree combinaisons VF/VR, VF/Pfnr_Down, Pfnr_Up/VR.'''
<p>'''VF2, VR, Pfnr_up, Pfnr_down are four oligos that we used for plasmid amplification. We used tree combinaisons VF/VR, VF/Pfnr_Down, Pfnr_Up/VR.'''
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'''If the promoter Pfnr insert PSB1C3 plasmid successfully, tree fragments with specific size will be amplified.''' </p>
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'''If the promoter Pndh* insert pSB1C3 plasmid successfully, tree fragments with specific size will be amplified.''' </p>
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'''
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Protocol'''
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COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.
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We resuspend each colony with 20µL of H2O.
Used quantities :  
Used quantities :  
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* Mix : (it was divided in tubes for 4 different colonies for each oligo combinaison with 23µL of mix in each tube)  
* Mix : (it was divided in tubes for 4 different colonies for each oligo combinaison with 23µL of mix in each tube)  
** VF or Pfnr_Up : 6µL
** VF or Pfnr_Up : 6µL
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** VR or Pfnr_Down or VR : 6µL
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** VR or Pfnr_Down : 6µL
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** dNTP : 6µL
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** dNTP 10mM : 6µL
** Buffer Dream Taq : 30µL  
** Buffer Dream Taq : 30µL  
** Dream Taq : 6µL
** Dream Taq : 6µL
** H2O : 246µL
** H2O : 246µL
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[[File:PsPCR2908.jpg|400px]]
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[[File:PSPCR0307.jpg|400px]]
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===='''2 - Culture of Bba_K1155000, Bba_I732017, Bba_K592009 '''====
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===='''2 - Culture of BBa_K1155000 '''====
Sheng
Sheng
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We made we culture by streaking 2 colonies of 07/02/13 culture of Bba_K1155000, Bba_I732017, Bba_K592009. We also did liquid culture of each one.   
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We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_K1155000. We also did liquid culture of each one. 
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We let culture at 37°C.
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We incubate culture at 37°C.
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===='''Objective : obtaining BBa_K1155003, BBa_K1155007'''====
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===='''1 - Culture of BBa_I732017, BBa_K592009 '''====
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Sheng
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{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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Tranformation of 07/02/13 works. We will do new cultures.
 +
|}
 +
 
 +
We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_I732017, BBa_K592009. We also did liquid culture of each one.   
 +
We incubate culture at 37°C.
 +
 
{| border="1" align="center"
{| border="1" align="center"

Latest revision as of 00:51, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 3

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155000

1 - Colony PCR of BBa_K1155000 to check good insertion of Pndh* in pSB1C3

Abdou, Sheng, Zhou

Tranformation of 07/02/13 works. We will do a PCR colony.

Colonies count for BBa_K1155000 :

  • Standard concentration :
    • BBa_K1155000 : 0 colony
  • High concentration :
    • BBa_K1155000 : 2 colonies

Ps0307jour.jpg


PSprimer07.jpg


Primers and PCR :

VF2, VR, Pfnr_up, Pfnr_down are four oligos that we used for plasmid amplification. We used tree combinaisons VF/VR, VF/Pfnr_Down, Pfnr_Up/VR. If the promoter Pndh* insert pSB1C3 plasmid successfully, tree fragments with specific size will be amplified.

Protocol

We resuspend each colony with 20µL of H2O.

Used quantities :

  • DNA : 2µL
  • Mix : (it was divided in tubes for 4 different colonies for each oligo combinaison with 23µL of mix in each tube)
    • VF or Pfnr_Up : 6µL
    • VR or Pfnr_Down : 6µL
    • dNTP 10mM : 6µL
    • Buffer Dream Taq : 30µL
    • Dream Taq : 6µL
    • H2O : 246µL

PSPCR0307.jpg

2 - Culture of BBa_K1155000

Sheng

We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_K1155000. We also did liquid culture of each one. We incubate culture at 37°C.

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Culture of BBa_I732017, BBa_K592009

Sheng

Tranformation of 07/02/13 works. We will do new cultures.

We made new cultures by streaking 2 colonies of 07/02/13 culture of BBa_I732017, BBa_K592009. We also did liquid culture of each one. We incubate culture at 37°C.


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