Team:Paris Saclay/Notebook/August/8

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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((((((((===='''Objective : obtaining FNR and BphR2 proteins'''====
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===='''Objective : obtaining FNR and BphR2 proteins'''====
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====1 - Gel purification of PCR product : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I====
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====1 - PCR product (made the 08/01/2013) purification====
 
Damir
Damir
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available quantity:
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Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
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* FNR Part1 : 10 µl
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* FNR Part2 : 19 µl
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* RBS FNR Part1 :16.1µl
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* RBS BphR2 Part1 : 28µl
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* BphR2 Part1 : 16.4 µl
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* BphR2 Part2 : 18.9 µl
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Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
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<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
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))))))))))))))))))))))))))))))
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{|
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
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{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 20:02, 16 September 2013

Navigation Home Team Project Labwork Notebook Human practices

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 was right

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 obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.

2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1

2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1

Anaïs

PsNBa8 eelution.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8% 
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.

2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel

Nadia

Protocol : Electroelution 

We let the plasmid precipitate during the night.))))))))))))))))))))))

Objective : obtaining Bba_K1155007

1 - Colony PCR of Bba_K115007 in DH5α

Anaïs

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 - Gel purification of PCR product : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Damir

Protocol : Gel purification

CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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