Paris/July 20

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< Paris(Difference between revisions)
(Glycerol stock)
 
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[[Paris/July 19|yesterday]] -- [[Paris/July 21|tomorrow]] <br>
==PCRs==
==PCRs==
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'''Je n’ai pas pu lancer la PCR car :
 
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Les oligos O10 et O11 ne sont pas dilués à 10μM (ni les autres oligos après O9 d’ailleurs ??!!). Les fiches des oligos ne sont pas dans le classeur, je ne peux donc pas savoir à quelle concentration est le stock !'''
 
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NB : Tous les oligos en stock sont à 100µM. Et ils sont ensuite dilués à 10µM dans l'autre boïte
 
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{{Paris_PCR_0| Title = Lox71-KmR-Lox66
{{Paris_PCR_0| Title = Lox71-KmR-Lox66
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|Annealing= 55°(3x)-63°(35x)C
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|Annealing= 55
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|Elongation= 2m00'
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|Elongation= 3m00'
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|Cycles= 38
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|Cycles= 35
|Buffer=  5x 10µL
|Buffer=  5x 10µL
|MgCl2= 10µM 0µL
|MgCl2= 10µM 0µL
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|n_oligoR= 11  Lox66-KmR-R
|n_oligoR= 11  Lox66-KmR-R
|v_oligoR= 2.5µL
|v_oligoR= 2.5µL
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|water= 30µL
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|water= 34µl
|pol= Phusion 0.5µL
|pol= Phusion 0.5µL
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|DNA= MP3_pSB1AK3-BBa_B0015 2µL dil.100
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|DNA= MP3_pSB1AK3-BBa_B0015 0.5µl
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|Size=
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|Size= 1084
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|Success=
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|Success= ?
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|Image=
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|Band=
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|}}
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{{Paris_PCR_0| Title = Lox71-FtsA-FtsZ
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|Annealing= 55
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|Elongation= 3m00'
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|Cycles= 35
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|Buffer=  5x 10µL
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|MgCl2= 10µM 0µL
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|dNTP= 10µM 1µL
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|n_oligoF= 3 Lox71-FtsA-F
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|v_oligoF= 2.5µL
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|n_oligoR= 2 FtsZ-R
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|v_oligoR= 2.5µL
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|water= 34µl
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|pol= Phusion 0.5µL
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|DNA= toothpick in MG1655 glycerol
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|Size= 2588
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|Success= ? The PCR seemed to have worked but the purification failed
|Image=
|Image=
|Band=
|Band=
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* BBa_pJ23107 clone 3&4
* BBa_pJ23107 clone 3&4
* BBa_I0500 clone 1&2
* BBa_I0500 clone 1&2
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== E.Coli pKS::DGAT ==
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We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015  on different LB medium (See [[Paris/July_18#E.coli_pKS::DGAT|July 18]]).
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 +
* Observations:
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We can observe colonies (X10).
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[[Image: coli_dgat_07202007.jpg|center|900px]]
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 +
* Interpretation
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 +
We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.

Latest revision as of 17:49, 7 October 2007

yesterday -- tomorrow

Contents

PCRs

PCR : Lox71-KmR-Lox66
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1084
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 10 Lox71-KmR-F 2.5µL  ?
3m00' Oligo R 10µM 11 Lox66-KmR-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA MP3_pSB1AK3-BBa_B0015 0.5µl
PCR : Lox71-FtsA-FtsZ
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2588
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL  ? The PCR seemed to have worked but the purification failed
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in MG1655 glycerol

Glycerol stock

  • BBa_pJ23107 clone 3&4 (S14)
  • BBa_I0500 clone 1&2 (S13)

Minipreps on Biobricks

  • BBa_pJ23107 clone 3&4
  • BBa_I0500 clone 1&2

E.Coli pKS::DGAT

We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015 on different LB medium (See July 18).

  • Observations:

We can observe colonies (X10).

Coli dgat 07202007.jpg
  • Interpretation

We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.