Team:Paris Saclay/Notebook/July/3

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{| border="1" align="center"
 
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|colonies
 
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|Normal concentration
 
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|High concentration
 
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|control
 
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|<center>11</center>
 
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|<center>60</center>
 
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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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<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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<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>
 
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<br>
 
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<p>So we had prepared 4(colonies)*3(amplification) = 12 PCR tubes. </p>
 
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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>
 
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<p>PCR programe</p>
 
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<br>
 
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<center>[[File:PSPCR0307.jpg|500px]]</center>
 
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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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<u>Culture confirmation</u>
<u>Culture confirmation</u>
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===='''2 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009'''====
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Revision as of 14:34, 22 September 2013

Culture confirmation

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.





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Contents

Notebook : July 1

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155000

1 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009

Abdou, Sheng, Zhou

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

Colonies count :

  • Standard concentration :
    • Bba_K1155000 : 0
  • High concentration :
    • Bba_K1155000 : 2


PSprimer07.jpg


Primer 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 Pfnr insert PSB1C3 plasmid successfully, tree fragments with specific size will be amplified.

COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.

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 or VR : 6µL
    • dNTP : 6µL
    • Buffer Dream Taq : 30µL
    • Dream Taq : 6µL
    • H2O : 246µL

PsPCR2908.jpg

2 - Colony PCR of Bba_K1155000, Bba_I732017, Bba_K592009

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