Team:Paris Saclay/Notebook/July/26

From 2013.igem.org

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(→2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP)
 
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : obtaining Bba_K1155003'''====
+
===='''Objective : obtaining BBa_K1155003'''====
-
===='''1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : RBS-AmilCP '''====
+
===='''1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''====
Abdou
Abdou
-
Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]]
+
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
We used 90µL of DNA.
We used 90µL of DNA.
 +
At the end, we mix our DNA with 20µL of water.
-
===='''2 - Ligation of RBS-AmilCP and Term-PSB1C3'''====
+
===='''2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP'''====
 +
 
 +
Abdou
 +
 
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel2607.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye
 +
* Well 2 : 6µL of DNA Ladder
 +
* Gel : 1%
 +
|}
 +
 
 +
Expected sizes :
 +
* RBS-AmilCP : 3500bp
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragments at the right size. We will ligate RBS-AmilCP with Term.
 +
|}
 +
 
 +
===='''3 - Ligation of RBS-AmilCP and Term-pSB1C3'''====
Xavier
Xavier
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* RBS-AmilCP : 2µL  
* RBS-AmilCP : 2µL  
-
* Term in PSB1C3 : 2.5µL
+
* Term in pSB1C3 : 2.5µL
* Buffer : 1µL
* Buffer : 1µL
* Ligase : 1µL
* Ligase : 1µL
* H2O :  3.5µL
* H2O :  3.5µL
-
===='''Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
===='''1 - Electrophoresis of PCR products : NarK, NarG, NirB'''====
===='''1 - Electrophoresis of PCR products : NarK, NarG, NirB'''====
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{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel12607.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 5µL of NirB+1µL of 6X loading dye
* Well 1 : 5µL of NirB+1µL of 6X loading dye
-
* Well 2 : 5µL of NirB+1µL of 6X loading dye LA MEME CHOSE ???????
+
* Well 2 : 5µL of NirB+1µL of 6X loading dye  
* Well 3 : 5µL of NarG+1µL of 6X loading dye
* Well 3 : 5µL of NarG+1µL of 6X loading dye
* Well 4 : 5µL of NarG+1µL of 6X loading dye
* Well 4 : 5µL of NarG+1µL of 6X loading dye
* Well 5 : 6µL DNA Ladder
* Well 5 : 6µL DNA Ladder
-
* Well 6 : 5µL of NarK+1µL of 6X loading dye NUMERO 4 ???????????????
+
* Well 6 : 5µL of NarK+1µL of 6X loading dye  
* Well 7 : 5µL of NarK+1µL of 6X loading dye
* Well 7 : 5µL of NarK+1µL of 6X loading dye
* Well 8 : 5µL of NarK+1µL of 6X loading dye  
* Well 8 : 5µL of NarK+1µL of 6X loading dye  
Line 50: Line 71:
Expected sizes :  
Expected sizes :  
-
* NarK, NarG, NirB : ...
+
* NarK, NarG, NirB : 200bp
{|
{|
Line 61: Line 82:
Abdou, Xavier
Abdou, Xavier
-
Used quantities : cf PCR précédente !!!!!!!!!!!!!
+
Used quantities :  
-
... ADN non dilué
+
 
 +
* NarK :
 +
** Buffer phusion : 10µL
 +
** dNTP : 1µL
 +
** Oligo 47 : 2µL
 +
** Oligo 48 : 2µL
 +
** Concentrated DNA : 2µL
 +
** Phusion : 0.25µL
 +
 +
* NarG :
 +
** Buffer phusion : 10µL
 +
** dNTP : 1µL
 +
** Oligo 49 : 2µL
 +
** Oligo 50 : 2µL
 +
** Concentrated DNA : 2µL
 +
** Phusion : 0.25µL
 +
 
 +
* NirB :
 +
** Buffer phusion : 10µL
 +
** dNTP : 1µL
 +
** Oligo 45 : 2µL
 +
** Oligo 46 : 2µL
 +
** Concentrated DNA : 2µL
 +
** Phusion : 0.25µL
 +
 
 +
PCR Program :
-
PCR program : cf PCR précédente !!!!!!!!!
+
[[File:PsPCR2507.jpg|400px]]
-
SCHEMA !!!!!!!!!!!!!!
+
===='''3 - Electrophoresis of PCR products : NarK, NarG, NirB'''====
===='''3 - Electrophoresis of PCR products : NarK, NarG, NirB'''====
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{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel22607.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
* Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
Line 85: Line 130:
Expected sizes :  
Expected sizes :  
-
* NarK, NarG, NirB : ...
+
* NarK, NarG, NirB : 200bp
{|
{|
Line 96: Line 141:
Xavier
Xavier
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
-
REPRIS dans 20µL !!!!!!!!
+
At the end we mix our DNA in 20µL of H2O.
===='''5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI'''====
===='''5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI'''====
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 +
==='''B - PCB sensor system'''===
 +
 +
===='''Objective : obtaining BBa_K1155002'''====
 +
 +
===='''1 - Tranformation of BBa_K1155002 in DH5α '''====
 +
 +
Abdou, Xavier
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial tranformation]]
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/July/25|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/July/30|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
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Latest revision as of 00:29, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP

Abdou

Protocol : Ethanol precipitation

We used 90µL of DNA. At the end, we mix our DNA with 20µL of water.

2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP

Abdou

Psgel2607.jpg
  • Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye
  • Well 2 : 6µL of DNA Ladder
  • Gel : 1%

Expected sizes :

  • RBS-AmilCP : 3500bp

We obtain fragments at the right size. We will ligate RBS-AmilCP with Term.

3 - Ligation of RBS-AmilCP and Term-pSB1C3

Xavier

Used quantities :

  • RBS-AmilCP : 2µL
  • Term in pSB1C3 : 2.5µL
  • Buffer : 1µL
  • Ligase : 1µL
  • H2O : 3.5µL

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou

Psgel12607.jpg
  • Well 1 : 5µL of NirB+1µL of 6X loading dye
  • Well 2 : 5µL of NirB+1µL of 6X loading dye
  • Well 3 : 5µL of NarG+1µL of 6X loading dye
  • Well 4 : 5µL of NarG+1µL of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of NarK+1µL of 6X loading dye
  • Well 7 : 5µL of NarK+1µL of 6X loading dye
  • Well 8 : 5µL of NarK+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • NarK, NarG, NirB : 200bp

We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos.

2 - PCR of NarK, NarG, NirB

Abdou, Xavier

Used quantities :

  • NarK :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 47 : 2µL
    • Oligo 48 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL
  • NarG :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 49 : 2µL
    • Oligo 50 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL
  • NirB :
    • Buffer phusion : 10µL
    • dNTP : 1µL
    • Oligo 45 : 2µL
    • Oligo 46 : 2µL
    • Concentrated DNA : 2µL
    • Phusion : 0.25µL

PCR Program :

PsPCR2507.jpg

3 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou, Xavier

Psgel22607.jpg
  • Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
  • Well 2 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
  • Well 3 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
  • Well 4 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
  • Well 5 : 6µL DNA Ladder
  • Well 6 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
  • Well 7 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
  • Gel : 1.2%

Expected sizes :

  • NarK, NarG, NirB : 200bp

We obtain fragments at the right size. We can purify it.

4 - Gel purification of PCR products : NarK, NarG, NirB

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

At the end we mix our DNA in 20µL of H2O.

5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI

Abdou

Quantities used :

  • NarK, NarG, NirB : 10µL
  • Buffer FD : 3µL
  • EcoRI FD : 1.5µL
  • PstI FD : 1.5µL
  • H2O : 14µL

We let the digestion at 37°C during 1h30 and then at -20°C.


B - PCB sensor system

Objective : obtaining BBa_K1155002

1 - Tranformation of BBa_K1155002 in DH5α

Abdou, Xavier

Protocol : Bacterial tranformation

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