Team:Paris/July 25

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== Extraction of the DNA ==
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== DNA Extraction ==
* Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
* Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
* Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))
* Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))
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* Test of the succeed of the extraction by electrophoresis on 2µl of the parts extracted.  
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* Analysis of the extraction success by electrophoresis on 2µl of the parts extracted.  
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-D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml
-D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml
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=== Determination of DNA concentration by electrophoresis ===
=== Determination of DNA concentration by electrophoresis ===
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==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments
==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments
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== Culture ==
== Culture ==
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in 10ml LB with adaptated antibiotics
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in 10ml LB with adaptated antibiotics, all the Biobricks used before (MP100 --> MP122)
* will be use to do minipreps
* will be use to do minipreps
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== MiniPreps ==
== MiniPreps ==
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|align="center"|[http://partsregistry.org/Part:BBa_E0840 E0840]  
|align="center"|[http://partsregistry.org/Part:BBa_E0840 E0840]  
|align="center"|gfp tri-part; strong rbs
|align="center"|gfp tri-part; strong rbs
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Latest revision as of 15:32, 31 July 2008

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Contents

DNA Extraction

  • Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
  • Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))
  • Analysis of the extraction success by electrophoresis on 2µl of the parts extracted.


Determination of DNA concentration by spectrophotometry

Conditions:


  • 2µl of the DNA extracted / 98µl of pure water
  • Reading against the adaptated blank (2µl of the elution's buffer / 98µl of pure water)


Results :


-D100 --> D110 cl-1 ES : Absorbance ~20-50µg/ml ; DO 260/280 = 1,5 ; DO 260/230 = 0,0-0.20

-D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml


Determination of DNA concentration by electrophoresis

  • to check for the samples from Qiagen's protocol, if the absence of detection of absorbance, is due to a problem of reading or a problem during the extraction, we realise an electrophoresis.


==> conclusion : we don't succeed to detect DNA by spectrophometry or electrophoresis for the samples produced by Qiagen's protocol.


==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments

Culture

in 10ml LB with adaptated antibiotics, all the Biobricks used before (MP100 --> MP122)

  • will be use to do minipreps


MiniPreps

  • Use of Promega's protocol on all the clones cultivated on the 24th.
  • Preparation of 50µl of minipreps in simple.


list of Minipreps

Name Biobrick Description
MP100 [http://partsregistry.org/Part:BBa_B0034 B0034] Strongest RBS (Efficiency = 1)
MP116 [http://partsregistry.org/Part:BBa_J23100 J23100] Strong constitutive promoter in J61002
MP120 [http://partsregistry.org/Part:BBa_B0030 B0030] Strong RBS (Efficiency = 0,6)
MP121 [http://partsregistry.org/Part:BBa_E0422 E0422] ECFP (RBS+LVA+Term)
MP122 [http://partsregistry.org/Part:BBa_E0840 E0840] gfp tri-part; strong rbs
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