Team:Paris Saclay/Notebook/August/1

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=='''Lab work'''==
=='''Lab work'''==
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o
 
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
-
===='''1 - Gibson assembly.'''====
 
-
* BphR2 part1, BphR2 part2 and plasmid PSB1C3
+
===='''Objective : obtaining FNR and BphR2 proteins'''====
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*_FNR_part1, FNR part1 and plasmid PSB1C3
+
-
* RBS FNR part1 and FNR_part2 and plasmid PSB1C3
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-
Protocol : [[Team:Paris_Saclay/Protocols/Gibson|PCR_clean_up]]
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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
 +
 +
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
-
Adding amount:
 
{|
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
The PCR products were good. Now we will do the Gibson assembly.
 +
|}
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| style="width:700px;border:1px solid black;vertical-align:top;" |
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===='''2 - Gibson assembly.'''====
-
*Epperdorf 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(2ul),Water(1ul),MIX Gibson (15 ul)
+
 
-
*Epperdorf 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(2ul),Water(1ul),MIX Gibson (15 ul)
+
Abdou, Xiaojing
-
*Epperdorf 3 : Bbs FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(2ul),Water(1ul),MIX Gibson (15 ul)
+
 
 +
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 :
 +
 
 +
[[File:PsPCRBSBphR23007.jpg|400px]]
 +
 
 +
===='''3 - Electrophoresis of the PCR products : RBS-BphR2 Part I'''====
 +
 
 +
Xavier, XiaoJing
 +
 
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel10108.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained our fragment at the right size. We will purify it.
|}
|}
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 +
{| border="1" align="center"
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|[[Team:Paris Saclay/Notebook/July/31|<big>Previous day</big>]]
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 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/2|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

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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