Team:Paris Saclay/Notebook/July/8

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : July 8'''=
='''Notebook : July 8'''=
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=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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==''Summary:''==
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===='''Objective : BBa_K1155003, BBa_K1155007'''====
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FNR regulator system:
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*performed a digestion for AmilCP and LacZ with Not I, Xho I, EcoR I and PST I.
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*electrophoresis for those digest products
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*enrichment culture for clone of the briobrick BBa_K1155000
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*electrophoresis for extracted products of last friday
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===='''1 - Digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
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=='''Lab work'''==
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Abou, Anaïs, Sheng, Zhou
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<u>Restriction digest</u>
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Used quantities :
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<br>
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<p>For some of the enzymes which are possible to use one common buffer, we did double digest
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* NotI :
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** BBa_K592009 : 2µL
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** Buffer orange : 2µL
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** NotI : 0.5µL
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** H2O : 15.5µL
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DNA enzyme buffer
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* XhoI :
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AmilCP1 Not I Orange
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** BBa_K592009 : 2µL
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AmilCP2 Not I Orange
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** Buffer red : 2µL
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AmilCP1 Xho I Red
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** XhoI : 0.5µL
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AmilCP2 Xho I Red
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** H2O : 15.5µL
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AmilCP1 EcoR I Orange
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AmilCP2 EcoR I Orange
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LacZ1 EcoR I + PST I Orange
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LacZ2 EcoR I + PST I Orange
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Volume added:
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* EcoRI :  
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DNA 2(µl)
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** BBa_K592009 : 2µL
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buffer 2(µl)
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** Buffer orange : 2µL
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Enzyme(foreach) 0.5(µl)
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** EcoRI : 0.5µL
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H20 15.5(µl)
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** H2O : 15.5µL
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total 20(µl)
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Notes: by using double digest, an informatics tool available in Fermentas web site, it is possible to use one buffer for both EcoR I and PST I.
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* EcoRI/PstI :  
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2. The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).
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** BBa_I732017 : 2µL
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** Buffer orange : 2µL
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** EcoRI : 0.5µL
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** PstI : 0.5µL
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** H2O : 15µL
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Volume added: for each well
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We let our digestion 1h30 at 37°C.
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plasmid 10µl
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OR Pfnr 5µl
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Blue
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Results:
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===='''2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
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Abdou, Anaïs, Sheng, Zhou
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{|
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| style="width:350px;border:1px solid black;" |[[File:Psgel0807.jpg|500px]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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* Well 1 : 6µL DNA Ladder
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* Well 2 : 5µL BBa_K592009 clone 1 digested by EcoRI+1µL of 6X loading dye
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* Well 3 : 5µL BBa_K592009 clone 2 digested by EcoRI+1µL of 6X loading dye
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* Well 4 : 5µL BBa_K592009 clone 1 digested by NotI+1µL of 6X loading dye
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* Well 5 : 5µL BBa_K592009 clone 2 digested by NotI+1µL of 6X loading dye
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* Well 6 : 5µL BBa_K592009 clone 1 digested by XhoI+1µL of 6X loading dye
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* Well 7 : 5µL BBa_K592009 clone 2 digested by XhoI+1µL of 6X loading dye
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* Well 8 : 5µL BBa_I732017 clone 1 digested by EcoRI/PstI+1µL of 6X loading dye
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* Well 9 : 5µL BBa_I732017 clone 2 digested by EcoRI/PstI+1µL of 6X loading dye
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* Well 10 : -
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* Well 11 : -
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* Well 12 : 6µL DNA Ladder
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* Gel : 1%
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|}
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3. Overnight incubation at 37°C with agitation.
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Expected sizes :
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4. The electrophoresis for the plasmids which we had extracted last week
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* BBa_K592009 digested by NotI : 2046bp + 693bp
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* BBa_K592009 digested by XhoI : 1842bp + 892bp
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* BBa_I732017 : 3093bp
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Volume added:  
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{|
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Pfnr2 R1P R2 A1P
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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10µl 10µl 10µl 10µl
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We obtain fragments at the right size. Our extraction was good.
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|}
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Migration at 100V for 25mins then continued till 45 minutes:
 
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Results:
 
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The concentration of Pfnr2 was detected by NanoDrop :
 
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C = 1788.8 ng/µl
 
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260/280 = 2.08
 
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We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.
 
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<br>
 
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Latest revision as of 23:55, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 8

Lab work

A - Aerobic/Anaerobic regulation system

Objective : BBa_K1155003, BBa_K1155007

1 - Digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI

Abou, Anaïs, Sheng, Zhou

Used quantities :

  • NotI :
    • BBa_K592009 : 2µL
    • Buffer orange : 2µL
    • NotI : 0.5µL
    • H2O : 15.5µL
  • XhoI :
    • BBa_K592009 : 2µL
    • Buffer red : 2µL
    • XhoI : 0.5µL
    • H2O : 15.5µL
  • EcoRI :
    • BBa_K592009 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • H2O : 15.5µL
  • EcoRI/PstI :
    • BBa_I732017 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • PstI : 0.5µL
    • H2O : 15µL

We let our digestion 1h30 at 37°C.

2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI

Abdou, Anaïs, Sheng, Zhou

Psgel0807.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL BBa_K592009 clone 1 digested by EcoRI+1µL of 6X loading dye
  • Well 3 : 5µL BBa_K592009 clone 2 digested by EcoRI+1µL of 6X loading dye
  • Well 4 : 5µL BBa_K592009 clone 1 digested by NotI+1µL of 6X loading dye
  • Well 5 : 5µL BBa_K592009 clone 2 digested by NotI+1µL of 6X loading dye
  • Well 6 : 5µL BBa_K592009 clone 1 digested by XhoI+1µL of 6X loading dye
  • Well 7 : 5µL BBa_K592009 clone 2 digested by XhoI+1µL of 6X loading dye
  • Well 8 : 5µL BBa_I732017 clone 1 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 9 : 5µL BBa_I732017 clone 2 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 10 : -
  • Well 11 : -
  • Well 12 : 6µL DNA Ladder
  • Gel : 1%

Expected sizes :

  • BBa_K592009 digested by NotI : 2046bp + 693bp
  • BBa_K592009 digested by XhoI : 1842bp + 892bp
  • BBa_I732017 : 3093bp

We obtain fragments at the right size. Our extraction was good.


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