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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 : 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 9: / Line 9: @@
 ===='''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 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
@@ Line 25: / Line 25: @@
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-The PCR was good. Now we will do the Gibson assembly.
+The PCR products were good. Now we will do the Gibson assembly.
 |}
@@ Line 35: / Line 35: @@
 * BphR2 :
-** PSB1C3 : 2µL
+** Gibson PCR of pSB1C3 : 2µL
 ** BphR2 Part I : 1µL
 ** BphR2 Part II : 1µL
@@ Line 42: / Line 42: @@
 * FNR :
-** PSB1C3 : 2µL
+** Gibson PCR of pSB1C3 : 2µL
 ** FNR Part I : 1µL
 ** FNR Part II : 1µL
@@ Line 49: / Line 49: @@
 * RBS-FNR :
-** PSB1C3 : 2µL
+** Gibson PCR of pSB1C3: 2µL
 ** RBS-FNR Part I : 1µL
 ** FNR Part II : 1µL
@@ Line 55: / Line 55: @@
 ** 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.
-===='''3 - PCR of : RBS-BphR2 Part I'''====
+===='''3 - PCR of RBS-BphR2 Part I'''====
 Abdou
@@ Line 66: / Line 66: @@
 ** Oligo 55R : 1µL
 ** Buffer phusion : 5µL
-** DNA : 0.25µL
+** DNA (''P. pseudoalcaligenes'' KF 707 genomic DNA) : 0.25µL
-** dNTP : 1µL
+** dNTP 10mM : 1µL
-** Phusion : 5µL
+** Enzyme Phusion : 5µL
 ** H2O : 36.5µL
@@ Line 80: / Line 80: @@
 {|
-| style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]]
+| 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
+* Well 2 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
 * Gel : 0.8%
 |}
 Expected sizes :
-* RBS-BphR2 Part I : ....
+* RBS-BphR2 Part I : 197pb
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-We obtain fragments at the right size. We will purify it.
+We obtained our fragment at the right size. We will purify it.
 |}

Team:Paris Saclay/Notebook/August/1

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