Paris/July 14

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[[Paris/July 13|yesterday]] -- [[Paris/July 15|tomorrow]] <br>
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'''Bastille's Day!!!'''
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[[Image:Toureiffel1.JPG|thumb|300px|]]
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[[Image:Toureiffel2.JPG|thumb|300px|]]
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[[Image:Toureiffel3.JPG|thumb|300px|]]
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<br>
== What to do ? ==
== What to do ? ==
Line 7: Line 16:
* Make a gel (0.8%) and migrate the PCR products
* Make a gel (0.8%) and migrate the PCR products
* Purify them
* Purify them
 +
* Do the assembly PCR
 +
If enough time (we can also wait to have the plasmids to do everything at the same time):
If enough time (we can also wait to have the plasmids to do everything at the same time):
* Digestion of the purifications products with appropriate enzymes
* Digestion of the purifications products with appropriate enzymes
* Purification
* Purification
 +
 +
 +
== What has been done ==
 +
 +
* Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):<br>
 +
- Isolation of 10 different colonies on LB Citrate Erythro DAP plates
 +
 +
* Seeding LB-Amp cultures of the following strains: <br>
 +
- DH5alpha transformed with the plasmid carrying the Biobrick pJ23100 <br>
 +
- DH5alpha transformed with the plasmid carrying the Biobrick B0015 <br>
 +
 +
*These two overnight cultures will allow us to perform (tomorrow):
 +
- Minipreps to isolate the plasmids carrying the biobricks <br>
 +
- Glycerol stocks of the transformed strains <br>
 +
 +
== DGAT expressing E.coli ==
 +
 +
After transformation of DH5alpha E.coli with pKS::DGAT plasmid, a transformant clone was grown overnight in LB Ampicilline medium.
 +
 +
The culture (using a toothpick) was deposited on several solid LB media for growth:
 +
<br> +/- IPTG (inducer of DGAT expression, dgat gene being carried by pKS::DGAT plasmid)
 +
<br> +/- Nile Red (fat detection dye)
 +
<br> +/- oleate at 2 different concentrations (0.5 and 2mM)
 +
 +
== PCRs ==
 +
These two first PCRs aims at removing the Pst1 site in DGAT gene.
 +
 +
{{Paris_PCR_0| Title = fw-DGAT1 rev-deltaPst1
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|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 27 forDGAT1
 +
|v_oligoF= 1µL
 +
|n_oligoR= 13 DPst1-DGAT-R
 +
|v_oligoR= 1µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= plasmid pKs::DGAT
 +
|Size=
 +
|Success=YES
 +
|Image=Paris_14_07-DGAT.jpg
 +
|Band=1
 +
|}}
 +
 +
{{Paris_PCR_0| Title = fw_deltaPst1 rev-DGAT1
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 12 fw_deltaPst1
 +
|v_oligoF= 1µL
 +
|n_oligoR= 28 RevDGAT1
 +
|v_oligoR= 1µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= plasmid pKs::DGAT
 +
|Size=
 +
|Success=YES
 +
|Image=Paris_14_07-DGAT.jpg
 +
|Band=2
 +
|}}
 +
 +
{{Paris_PCR_0| Title = Lox71-FtsA-FtsZ-1
 +
|Name= Lox71-FtsA-FtsZ-1
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 2.5µL
 +
|n_oligoR= 4 DEcoR1-FtsZ-R
 +
|v_oligoR= 2.5µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= toothpick in glycerol stock of MG1655
 +
|Size=
 +
|Success=YES
 +
|Image=Paris_15_07-Gel2.jpg
 +
|Band=1-2
 +
|}}
 +
 +
 +
{{Paris_PCR_0| Title = FtsZ-2
 +
|Name= FtsZ-2
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 5 DEcoR1-FtsZ-F
 +
|v_oligoF= 10µM 2.5µL
 +
|n_oligoR= 2 FtsZ-R
 +
|v_oligoR= 10µM 2.5µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= toothpick in glycerol stock of MG1655
 +
|Size=
 +
|Success=YES
 +
|Image=Paris_15_07-Gel2.jpg
 +
|Band=3-4
 +
|}}
 +
 +
{{Paris_PCR_0| Title = Lox66-DapAColi
 +
|Name= Lox66-DapAColi
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 6 Lox66-DapAColi-F
 +
|v_oligoF= 10µM 2.5µL
 +
|n_oligoR= 7 DapAColi-R
 +
|v_oligoR= 10µM 2.5µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= toothpick in glycerol stock of MG1655
 +
|Size=
 +
|Success= No
 +
|Image=Paris_15_07-Gel2.jpg
 +
|Band=5-6
 +
|}}

Latest revision as of 17:47, 7 October 2007

yesterday -- tomorrow
Bastille's Day!!!

Toureiffel1.JPG
Toureiffel2.JPG
Toureiffel3.JPG



Contents

What to do ?

  • Remove the plate of the transduction experiment and the transformation ones from the 37°C incubator (Upper-left shelf): If it has worked and if someones comes on Sunday:
    • isolation of the clones from the transduction on petri-dishes (LB+DAP+Erm+Citrate)
    • LB + antibio culture of the transformants (to make a glycerol stock and to do MiniPreps)
  • Make a gel (0.8%) and migrate the PCR products
  • Purify them
  • Do the assembly PCR

If enough time (we can also wait to have the plasmids to do everything at the same time):

  • Digestion of the purifications products with appropriate enzymes
  • Purification


What has been done

  • Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):

- Isolation of 10 different colonies on LB Citrate Erythro DAP plates

  • Seeding LB-Amp cultures of the following strains:

- DH5alpha transformed with the plasmid carrying the Biobrick pJ23100
- DH5alpha transformed with the plasmid carrying the Biobrick B0015

  • These two overnight cultures will allow us to perform (tomorrow):

- Minipreps to isolate the plasmids carrying the biobricks
- Glycerol stocks of the transformed strains

DGAT expressing E.coli

After transformation of DH5alpha E.coli with pKS::DGAT plasmid, a transformant clone was grown overnight in LB Ampicilline medium.

The culture (using a toothpick) was deposited on several solid LB media for growth:
+/- IPTG (inducer of DGAT expression, dgat gene being carried by pKS::DGAT plasmid)
+/- Nile Red (fat detection dye)
+/- oleate at 2 different concentrations (0.5 and 2mM)

PCRs

These two first PCRs aims at removing the Pst1 site in DGAT gene.

PCR : fw-DGAT1 rev-deltaPst1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 27 forDGAT1 1µL YES
2m00' Oligo R 10µM 13 DPst1-DGAT-R 1µL Image (click to enlarge)
Number cycles Water 34µL Paris 14 07-DGAT.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA plasmid pKs::DGAT 1
PCR : fw_deltaPst1 rev-DGAT1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 12 fw_deltaPst1 1µL YES
2m00' Oligo R 10µM 28 RevDGAT1 1µL Image (click to enlarge)
Number cycles Water 34µL Paris 14 07-DGAT.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA plasmid pKs::DGAT 2
PCR : Lox71-FtsA-FtsZ-1
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL YES
2m00' Oligo R 10µM 4 DEcoR1-FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 1-2


PCR : FtsZ-2
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 5 DEcoR1-FtsZ-F 10µM 2.5µL YES
2m00' Oligo R 10µM 2 FtsZ-R 10µM 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 3-4
PCR : Lox66-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL
55°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 6 Lox66-DapAColi-F 10µM 2.5µL No
2m00' Oligo R 10µM 7 DapAColi-R 10µM 2.5µL Image (click to enlarge)
Number cycles Water 34µL Paris 15 07-Gel2.jpg
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in glycerol stock of MG1655 5-6
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