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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==''Summary:''==
 
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FNR regulator system:
 
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*performed another digestion for AmilCP, Pfnr plus sequence 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
 
=='''Lab work'''==
=='''Lab work'''==
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<u>Restriction digest</u>
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==='''A - Aerobic/Anaerobic regulation system'''===
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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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===='''Objective : BBa_K1155003, BBa_K1155007'''====
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{| border="1" align="center"
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|-
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|DNA
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|enzyme
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|buffer
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|-
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|AmilCP1
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|Not I
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|Orange
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-
|-
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|AmilCP2
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|Not I
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|Orange
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-
|-
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|AmilCP1
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|Xho I
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|Red
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|-
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|AmilCP2
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|Xho I
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|Red
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|-
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|AmilCP1
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|EcoR I
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|Orange
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|-
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|AmilCP2
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|EcoR I
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|Orange
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|-
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|LacZ1
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|EcoR I + PST I
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|Orange
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|-
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|LacZ2
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|EcoR I + PST I
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|Orange
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|}
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<br>
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<div>Volume added:</div>
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{| border="1" align="center"
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-
|-
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|DNA
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|2(µl)
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|-
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|buffer
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|2(µl)
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|-
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|Enzyme(foreach)
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|0.5(µl)
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|-
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|H20
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|15.5(µl)
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|-
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|total
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|20(µl)
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|}
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<br>
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===='''1 - Digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
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The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).
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Abou, Anaïs, Sheng, Zhou
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Results:
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Used quantities :  
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{| align="center"
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* NotI :  
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| style="width:350px;border:1px solid black;" | [[File:PS080713dig.jpg|center|350px]]
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** BBa_K592009 : 2µL
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| style="width:350px;border:1px solid black;" |
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** Buffer orange : 2µL
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*Well 1,12 : marker
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** NotI : 0.5µL
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*Well 2: AmilCP1 + Ecor I
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** H2O : 15.5µL
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*Well 3: AmilCP2 + Ecor I
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*Well 4: AmilCp1 + Not I
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*Well 5: AmilCP2 + Not I
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*Well 6: AmilCp1 + Xho I
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*Well 7: AmilCp1 + Xho I
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*Well 8: LacZ 1 + Ecor I + Pst I
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*Well 9: LacZ 2 + Ecor I + Pst I
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*Well 10: Nothing
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*Well 11: fnr promoter
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*gel 0.8%
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|}<br>
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* XhoI :
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** BBa_K592009 : 2µL
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** Buffer red : 2µL
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** XhoI : 0.5µL
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** H2O : 15.5µL
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* EcoRI :
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** BBa_K592009 : 2µL
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** Buffer orange : 2µL
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** EcoRI : 0.5µL
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** H2O : 15.5µL
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Overnight incubation at 37°C with agitation.
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* EcoRI/PstI :
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<br>
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** BBa_I732017 : 2µL
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The electrophoresis for the plasmids which we had extracted last week plus 3 other BioBrick promoter Bph R1, promoter Bph A1, Bph R2.
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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:
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We let our digestion 1h30 at 37°C.
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*Pfnr2: 10µl
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*R1P: 10µl
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*R2: 10µl
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*A1P: 10µl
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-
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Migration at 100V for 25mins then continued till 45 minutes:
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===='''2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
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<br>
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Results:
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{| align="center"
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Abdou, Anaïs, Sheng, Zhou
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| style="width:350px;border:1px solid black;" | [[File:PS08071345.jpg|right|350px]]
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| style="width:350px;border:1px solid black;" |
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*Well 1 : R2
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*Well 2 : R1P
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*Well 3 : A1P
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*Well 4 : Pfnr
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*gel 0.8%
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|}<br>
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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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{|
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*260/280 = 2.08
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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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We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.
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Expected sizes :
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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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{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragments at the right size. Our extraction was good.
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|}
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<u>Electrophoresis band size estimation</u><br>
 
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<p>We used Clonemanager for band size estimation:</p>
 
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<br>
 
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{| border="1" align="center"
 
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|-
 
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|Molecule
 
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|enzymes(Buffer)
 
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|Size
 
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|-
 
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|AmilCP clone
 
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|Nod I(Orange)
 
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|2046+693bp
 
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|-
 
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|AmilCP clone
 
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|XHOI(Red)
 
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|1842+892bp
 
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|-
 
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|BBa_K1155000
 
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|XHOI(Red)
 
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|1289+892bp
 
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|-
 
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|BBa_K1155000
 
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|ECoR I(Orange)
 
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|2151bp
 
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|}<br>
 
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<br>
 
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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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