Team:Paris Saclay/Notebook/August/1

From 2013.igem.org

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(3 - Electrophoresis of the PCR products : RBS-BphR2 Part I)
 
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===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
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===='''1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I PSB1C3 '''====
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===='''1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I in plasmid pSB1C3 '''====
Xavier
Xavier
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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The PCR was good. Now we will do the Gibson assembly.
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The PCR products were good. Now we will do the Gibson assembly.
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* BphR2 :  
* BphR2 :  
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** PSB1C3 : 2µL
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** Gibson PCR of pSB1C3 : 2µL
** BphR2 Part I : 1µL  
** BphR2 Part I : 1µL  
** BphR2 Part II : 1µL  
** BphR2 Part II : 1µL  
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* FNR :  
* FNR :  
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** PSB1C3 : 2µL
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** Gibson PCR of pSB1C3 : 2µL
** FNR Part I : 1µL
** FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
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* RBS-FNR :
* RBS-FNR :
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** PSB1C3 : 2µL
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** Gibson PCR of pSB1C3: 2µL
** RBS-FNR Part I : 1µL
** RBS-FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
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** H2O : 1µL
** H2O : 1µL
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We let these mix at 50°C during 1h.
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We incubated these Gibson assembly mixes at 50°C during 1h inside PCR machine.
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===='''3 - PCR of : RBS-BphR2 Part I'''====
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===='''3 - PCR of RBS-BphR2 Part I'''====
Abdou  
Abdou  
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** Oligo 55R : 1µL
** Oligo 55R : 1µL
** Buffer phusion : 5µL
** Buffer phusion : 5µL
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** DNA : 0.25µL
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** DNA (''P. pseudoalcaligenes'' KF 707 genomic DNA) : 0.25µL
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** dNTP : 1µL
+
** dNTP 10mM : 1µL
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** Phusion : 5µL  
+
** Enzyme Phusion : 5µL  
** H2O : 36.5µL
** H2O : 36.5µL
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{|
{|
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| style="width:350px;border:1px solid black;" |[[File:Psgel10108.jpg| 400px]]
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| style="width:350px;border:1px solid black;" |[[File:Psgel10108.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL of DNA Ladder
* Well 1 : 6µL of DNA Ladder
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* Well 2 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
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* Well 2 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
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|}  
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain fragments at the right size. We will purify it.
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We obtained our fragment at the right size. We will purify it.
|}
|}

Latest revision as of 17:08, 3 October 2013

Contents

Notebook : August 1

Lab work

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

Objective : obtaining FNR and BphR2 proteins

1 - Gel purification of PCR products : BphR2 Part I, BphR2 Part II, FNR Part I, FNR Part II, RBS-FNR Part I in plasmid pSB1C3

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • BphR2 Part I : 44 ng/µL
  • BphR2 Part II : 64 ng/µL
  • FNR Part I : 147 ng/µL
  • FNR Part II : 140 ng/µL
  • RBS-FNR Part I : 167 ng/µL
  • PSB1C3 : 159 ng/µL

The PCR products were good. Now we will do the Gibson assembly.

2 - Gibson assembly.

Abdou, Xiaojing

Used quantities :

  • BphR2 :
    • Gibson PCR of pSB1C3 : 2µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
    • H2O : 1µL
  • FNR :
    • Gibson PCR of pSB1C3 : 2µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
    • H2O : 1µL
  • RBS-FNR :
    • Gibson PCR of pSB1C3: 2µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL
    • H2O : 1µL

We incubated these Gibson assembly mixes at 50°C during 1h inside PCR machine.

3 - PCR of RBS-BphR2 Part I

Abdou

Used quantities :

    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer phusion : 5µL
    • DNA (P. pseudoalcaligenes KF 707 genomic DNA) : 0.25µL
    • dNTP 10mM : 1µL
    • Enzyme Phusion : 5µL
    • H2O : 36.5µL

PCR Program :

PsPCRBSBphR23007.jpg

3 - Electrophoresis of the PCR products : RBS-BphR2 Part I

Xavier, XiaoJing

Psgel10108.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-BphR2 Part I : 197pb

We obtained our fragment at the right size. We will purify it.


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