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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 :

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

Xavier, XiaoJing

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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@@ Line 4: / Line 4: @@
 =='''Lab work'''==
 ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
-===='''1 - Gibson assembly.'''====
-Andy,Xiaojing
-* BphR2_part1, BphR2_part2 and plasmid PSB1C3
+===='''Objective : obtaining FNR and BphR2 proteins'''====
-* FNR_part1, FNR part1 and plasmid PSB1C3
-* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
+===='''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
-Sample Volume:
 {|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+The PCR products were good. Now we will do the Gibson assembly.
+|}
-| style="width:700px;border:1px solid black;vertical-align:top;" |
+===='''2 - Gibson assembly.'''====
-*Tube 1 : BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(2ul),Water(1ul),MIX Gibson (15 ul)
-*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(2ul),Water(1ul),MIX Gibson (15 ul)
+Abdou, Xiaojing
-*Tube 3 : RBS_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.
 |}
-Put those 3 tubes into thermocycler at50℃ for 1 hour.
+{| border="1" align="center"
+|[[Team:Paris Saclay/Notebook/July/31|<big>Previous day</big>]]
+|[[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/Notebook/August/1

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