Team:Paris Saclay/Notebook/August/8

From 2013.igem.org

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*GFP : 1069kb
*GFP : 1069kb
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We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
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====2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1====
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We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.
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''2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1''
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====2 - Electrophoresis of Bba_J004450 digested by EcoRI/PstI====
Anaïs
Anaïs
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We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.
We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.
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''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
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==== 3- Electroelution of PSB3K3 digested by EcoRI/PstI====
Nadia
Nadia
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We let the plasmid precipitate during the night.
We let the plasmid precipitate during the night.
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===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
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===='''1 - Tranduction of Km in MG1655Z1 ====
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Anaïs, Nadia
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We didn't obtain colonies from the transduction of 08/07/13. We will do it again.
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Protocol : [[Team:Paris_Saclay/Protocols/transduction|Transduction]]
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Our Mutant bacteria was called BW : Δfnr::Km.
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Our wild type bacteria was called MG1655Z1.
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We did the first step of the protocol : bacteriophage stock which packed Km gene. 
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===='''Objective : obtaining Bba_K1155007'''====
===='''Objective : obtaining Bba_K1155007'''====

Revision as of 14:34, 28 September 2013

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Contents

Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the PSB3K3 backbone plasmid

1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion

Damir, Nadia

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8%

Expected sizes :

  • PSB3K3 : 2750kb
  • GFP : 1069kb

We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.

2 - Electrophoresis of Bba_J004450 digested by EcoRI/PstI

Anaïs

PsNBa8 eelution.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8%

We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.

3- Electroelution of PSB3K3 digested by EcoRI/PstI

Nadia

Protocol : Electroelution

We let the plasmid precipitate during the night.

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Tranduction of Km in MG1655Z1

Anaïs, Nadia

We didn't obtain colonies from the transduction of 08/07/13. We will do it again.

Protocol : Transduction

Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1.

We did the first step of the protocol : bacteriophage stock which packed Km gene.


Objective : obtaining Bba_K1155007

1 - Colony PCR of Bba_K115007 in DH5α

Anaïs

Tranformation of 08/07/13 works. we will make a PCR Colony.

Colonie repiquée dans 10µL d'eau pour chaque tube ???????

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 25 tubes for each promotor with 23µL of mix in each on)
    • Oligo ... : 3.5µL
    • Oligo ... : 3.5µL
    • Buffer Dream Taq : 70µL
    • dNTP : 28µL
    • Dream Taq : 5µL
    • H2O : 590µL

PCR Program :

PsPcr808.jpg

2 - Electrophoresis to check the colony PCR products : Bba_K1155007

Anaïs, Damir

350px
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 24 : 10µL of Bba_K1155007+2µL of 6X loading dye
  • Well 25 : 6µL DNA Ladder
  • Well 26 : 6µL DNA Ladder
  • Well 27 : 10µL of Bba_K1155007+2µL of 6X loading dye
  • Gel : 0.8%

Expected size :

  • Bba_K1155007 : 3583 bp

We obtain fragment at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007.


A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - Extraction of plasmid of BphR2, FNR, RBS-FNR

Damir

Transformation of 08/02/13 works. We will extract plasmids.

Protocol : Gel purification

We lost our plasmids. We will do the Gibson assembly again.

2 - Gel purification of RBS-BphR2 Part I

Nadia, XiaoJing

Protocol : Gel purification

We lost fragment. We will do the PCR again.


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