Team:Paris Saclay/Notebook/July/8

From 2013.igem.org

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We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.  
We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.  
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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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Revision as of 14:22, 26 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : July 8

Summary:

FNR regulator system:

  • performed another digestion for AmilCP, Pfnr plus sequence LacZ with Not I, Xho I, EcoR I and PST I.
  • electrophoresis for those digest products
  • enrichment culture for clone of the briobrick BBa_K1155000
  • electrophoresis for extracted products of last friday

Lab work

Restriction digest

For some of the enzymes which are possible to use one common buffer, we did double digest

DNA enzyme buffer
AmilCP1 Not I Orange
AmilCP2 Not I Orange
AmilCP1 Xho I Red
AmilCP2 Xho I Red
AmilCP1 EcoR I Orange
AmilCP2 EcoR I Orange
LacZ1 EcoR I + PST I Orange
LacZ2 EcoR I + PST I Orange


Volume added:
DNA 2(µl)
buffer 2(µl)
Enzyme(foreach) 0.5(µl)
H20 15.5(µl)
total 20(µl)


The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).

Results:

PS080713dig.jpg
  • Well 1,12 : marker
  • Well 2: AmilCP1 + Ecor I
  • Well 3: AmilCP2 + Ecor I
  • Well 4: AmilCp1 + Not I
  • Well 5: AmilCP2 + Not I
  • Well 6: AmilCp1 + Xho I
  • Well 7: AmilCp1 + Xho I
  • Well 8: LacZ 1 + Ecor I + Pst I
  • Well 9: LacZ 2 + Ecor I + Pst I
  • Well 10: Nothing
  • Well 11: fnr promoter
  • gel 0.8%


Overnight incubation at 37°C with agitation.
The electrophoresis for the plasmids which we had extracted last week plus 3 other BioBrick promoter Bph R1, promoter Bph A1, Bph R2.

Volume added:

  • Pfnr2: 10µl
  • R1P: 10µl
  • R2: 10µl
  • A1P: 10µl


Migration at 100V for 25mins then continued till 45 minutes:
Results:

PS08071345.jpg
  • Well 1 : R2
  • Well 2 : R1P
  • Well 3 : A1P
  • Well 4 : Pfnr
  • gel 0.8%


The concentration of Pfnr2 was detected by NanoDrop :

  • C = 1788.8 ng/µl
  • 260/280 = 2.08

We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.








Electrophoresis band size estimation

<p>We used Clonemanager for band size estimation:


Molecule enzymes(Buffer) Size
AmilCP clone Nod I(Orange) 2046+693bp
AmilCP clone XHOI(Red) 1842+892bp
BBa_K1155000 XHOI(Red) 1289+892bp
BBa_K1155000 ECoR I(Orange) 2151bp










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