Team:Paris Saclay/Notebook/July/3

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : July 3'''=
='''Notebook : July 3'''=
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=='''Lab work'''==
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=='''Summary:'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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FNR regulator system:
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*Continued what we started yesterday. Observed the Petri dish, selected the colonies. 4 colonies in total, they were 2 include FNR+plasmid in PSB1C3 and 2 from the control.
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===='''Objective : obtaining BBa_K1155000'''====
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*Used 4 primers: VF2, VR, Pfnr_up, Pfnr_down for the verification test. They were designed to cut 4 special sites for creating 3 different regions on plasmid chain: VF2/VR, VF2/PFNR_down, PFNR_up/VR. After the amplification, those PCR products had been put on electrophoresis gel for the verification.
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=='''Lab work'''==
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===='''1 - Colony PCR of BBa_K1155000 to check good insertion of Pndh* in pSB1C3'''====
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*A.aero/anaerobic regulation system
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Abdou, Sheng, Zhou
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:*2.BioBrick RBS+LacZ+terminator in plasmid PSB1C3
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:*BioBrick RBS+amilCP+terminator in plasmid PSB1C3
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<u>Observation</u>
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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 a PCR colony.
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|}
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<p>After the incubation overnight, we observed the Petri dishes. </p>
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Colonies count for BBa_K1155000 :
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<br>
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{| border="1" align="center"
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* Standard concentration :
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|-
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** BBa_K1155000 : 0 colony
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|colonies
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|Normal concentration
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|High concentration
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|-
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|control
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|<center>11</center>
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|<center>60</center>
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|-
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|Fnr in plasmid PSB1C3
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|<center>0</center>
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|<center>2</center>
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|}
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<br>
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<p>We picked up 4 colonies for further test (2 include FNR+plasmid in PSB1C3 and 2 from the control)</p>
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<u>Primer and PCR</u>
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* High concentration :
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** BBa_K1155000 : 2 colonies
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[[File:Ps0307jour.jpg|300px]]
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<p>VF2, VR, Pfnr_up, Pfnr_down are 4 primes that we used for plasmid restriction. The primers, if the promoter fnr entre the plasmid successfully will amplify 3 sub-pieces with specific size. They are VF/VR, VF/Pfnr_down, Pfnr_up/VR</p><br>
 
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<center>[[File:PSprimer07.jpg|300px]]</center>
 
<br>
<br>
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<p>So we had prepared 4(colonies)*3(amplification) = 12 PCR tubes. </p>
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<center>[[File:PSprimer07.jpg|right|250px]]</center>
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*Dream Taq(5µg/µl):2µl<br>
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*Buffer (Dream Taq) 10X:10µl<br>
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*dNTP:2µl<br>
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*Primer (F/R;F/fnr_R;fnr_F/R):2µl+2µl<br>
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*H2O:82µl<br>
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*Total:100µl (volume for 4 tubes, so 25µl for each)<br>
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<br>
<br>
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<p>PCR was programed for 95°C for 3mins and 30 cycles of 94°C 30s + 58°C 30s + 72°C 30s.</p>
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'''[https://2013.igem.org/Team:Paris_Saclay/Primers Primers] and PCR :'''
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<p>The PCR products was put on a gel of agarose 1.5% with BET (1.5%), migrated during 30 min at 100V. the picture analysis was be delayed to the next day.</p>
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<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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<br>
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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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<u>Culture confirmation</u>
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'''
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<br>
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Protocol'''
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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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We resuspend each colony with 20µL of H2O.
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Used quantities :
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* DNA : 2µL
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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)
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** VF or Pfnr_Up : 6µL
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** VR or Pfnr_Down : 6µL
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** dNTP 10mM : 6µL
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** Buffer Dream Taq : 30µL
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** Dream Taq : 6µL
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** H2O : 246µL
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[[File:PSPCR0307.jpg|400px]]
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===='''2 - Culture of BBa_K1155000 '''====
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Sheng
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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 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.
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|}
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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. 
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We incubate culture at 37°C.
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<br>
 
{| border="1" align="center"
{| border="1" align="center"
|[[Team:Paris Saclay/Notebook/July/2|<big>Previous day</big>]]
|[[Team:Paris Saclay/Notebook/July/2|<big>Previous day</big>]]

Latest revision as of 00:51, 5 October 2013

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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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