Team:Paris Saclay/Notebook/August/1

From 2013.igem.org

(Difference between revisions)
(→3 - Electrophoresis of the PCR products : RBS-BphR2 Part I)
 
(15 intermediate revisions not shown)
Line 9: Line 9:
===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''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
Xavier
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :  
Nanodrop :  
Line 25: Line 25:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
+
The PCR products were good. Now we will do the Gibson assembly.
|}
|}
Line 35: Line 35:
* BphR2 :  
* BphR2 :  
-
** PSB1C3 : 2µL
+
** Gibson PCR of pSB1C3 : 2µL
** BphR2 Part I : 1µL  
** BphR2 Part I : 1µL  
** BphR2 Part II : 1µL  
** BphR2 Part II : 1µL  
-
** Gibson mix : 15µL ???????????????
+
** Gibson mix : 15µL  
** H2O : 1µL
** H2O : 1µL
* FNR :  
* FNR :  
-
** PSB1C3 : 2µL
+
** Gibson PCR of pSB1C3 : 2µL
** FNR Part I : 1µL
** FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
-
** Gisbon mix : 15µL ???????????????
+
** Gisbon mix : 15µL  
** H2O : 1µL
** H2O : 1µL
* RBS-FNR :
* RBS-FNR :
-
** PSB1C3 : 2µL
+
** 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.
-
===='''3 - PCR of : RBS-BphR2 Part I'''====
+
===='''3 - PCR of RBS-BphR2 Part I'''====
Abdou  
Abdou  
Line 66: Line 66:
** Oligo 55R : 1µL
** Oligo 55R : 1µL
** Buffer phusion : 5µL
** Buffer phusion : 5µL
-
** 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
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;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL of DNA Ladder
* 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%
* Gel : 0.8%
|}  
|}  
Expected sizes :  
Expected sizes :  
-
* RBS-BphR2 Part I : ....
+
* 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.
 +
|}
 +
 
 +
 
 +
{| 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/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.


Previous day Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/1"