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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Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :  
Nanodrop :  
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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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CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
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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
-
** Gisbon mix : 15µL ???????????????
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** Gisbon mix : 15µL  
** H2O : 1µL
** H2O : 1µL
* 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
-
** Gibson mix : 15µL ????????????
+
** Gibson mix : 15µL  
** H2O : 1µL
** H2O : 1µL
-
We let these mix at 50°C during 1h.
+
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'''====
+
===='''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
+
** DNA (''P. pseudoalcaligenes'' KF 707 genomic DNA) : 0.25µL
-
** dNTP : 1µL
+
** dNTP 10mM : 1µL
-
** Phusion : ... µL 5 ?????????
+
** 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:Ps.jpg|350px]]
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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%
|}  
|}  
Expected sizes :  
Expected sizes :  
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* RBS-BphR2 Part I : ....
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* RBS-BphR2 Part I : 197pb
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
+
We obtained our fragment at the right size. We will purify it.
|}
|}
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===='''4 - Gel purification of PCR of : RBS-BphR2 Part I'''====
 
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Latest revision as of 17:08, 3 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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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