Team:Paris Saclay/Notebook/July/30

From 2013.igem.org

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(→5 - Gel purification of digestion of BBa_I732017 by EcoRI/PstI)
 
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
===='''1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB '''====
+
===='''1 - Inactivation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB '''====
Xavier
Xavier
Line 17: Line 17:
We used 30µL of DNA.
We used 30µL of DNA.
-
===='''Objective : obtaining Bba_K1155003'''====
+
===='''Objective : obtaining BBa_K1155003, BBa_K1155007'''====
-
===='''1 - Tranformation of Bba_K1155003 in DH5α '''====
+
===='''1 - Tranformation of BBa_K1155003 in DH5α '''====
Abdou
Abdou
Line 25: Line 25:
Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]]
Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]]
 +
===='''2 - Digestion of BBa_I732017 by EcoRI/PstI'''====
 +
 +
Zhou
 +
 +
Used quantities :
 +
* BBa_I732017 : 10µL
 +
* Buffer FD : 3µL
 +
* PstI FD : 1.5µL
 +
* EcoRI FD : 1.5µL
 +
* H2O : 14µL
 +
 +
We let the digestion 1h30 at 37°C.
 +
 +
===='''3 - Digestion of BBa_I732017 by EcoRI/SpeI'''====
 +
 +
Abdou
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
We used wrong enzymes so we will do the digestion again.
 +
|}
 +
 +
Used quantities :
 +
* BBa_I732017 : 10µL
 +
* Buffer FD : 2µL
 +
* SpeI FD : 1µL
 +
* EcoRI FD : 1µL
 +
* H2O : 6µL
 +
 +
We let the digestion 1h30 at 37°C.
 +
 +
===='''4 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI'''====
 +
 +
Zhou
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel23007.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 10µL of DNA Ladder
 +
* Well 2 : 10µL of BBa_I732017 digested by EcoRI/SpeI+4µL of 6X loading dye
 +
* Well 3 : 10µL of BBa_I732017 digested by EcoRI/SpeI+4µL of 6X loading dye
 +
* Gel : 1%
 +
|}
 +
 +
Expected size :
 +
* RBS-LacZ : 3090bp
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragments at the right size. We will purify it.
 +
|}
 +
 +
===='''5 - Gel purification of digestion of BBa_I732017 by EcoRI/SpeI'''====
 +
 +
Xavier
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost our fragments. We will do the digestion again.
 +
|}
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
Line 91: Line 153:
* PSB1C3 :  
* PSB1C3 :  
-
** Prefixe ... : 4µL
+
** Prefixe 51 : 4µL
-
** Suffixe ... : 4µL
+
** Suffixe 52 : 4µL
** Buffer Dream Taq : 40µL
** Buffer Dream Taq : 40µL
-
** DNA ... : 2µL
+
** DNA : 2µL
** dNTP : 4µL
** dNTP : 4µL
** Dream Taq : 1µL
** Dream Taq : 1µL
Line 108: Line 170:
[[File:PsPCRFNR3007.jpg|400px]]
[[File:PsPCRFNR3007.jpg|400px]]
-
 
-
* PSB1C3 :
 
-
 
-
[[File:Ps.jpg|400px]]
 
===='''2 - Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I  '''====
===='''2 - Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I  '''====
{|
{|
-
| style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel13007.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
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* FNR Part II : 200 kb
* FNR Part II : 200 kb
* RBS-FNR Part I : 615 kb
* RBS-FNR Part I : 615 kb
-
* PSB1C3 : ...
+
* PSB1C3 : 2070bp
{|
{|
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** Oligo 54F : 4µL
** Oligo 54F : 4µL
** Oligo 55R : 4µL
** Oligo 55R : 4µL
-
** Buffer .... : 40µL
+
** Buffer Dream Taq : 40µL
** DNA : 2µL
** DNA : 2µL
** dNTP : 4µL
** dNTP : 4µL
-
** .... : 1µL
+
** Dream Taq : 1µL
** H2O : 146µL
** H2O : 146µL
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===='''4 - Gel purification of PCR of : RBS-BphR2 Part I'''====
===='''4 - Gel purification of PCR of : RBS-BphR2 Part I'''====
-
????????
+
Xavier
 +
 
 +
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost our fragments. We will do the PCR again.
 +
|}
 +
 
Line 174: Line 240:
Abdou, Xavier
Abdou, Xavier
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
The transformation was good. We will make a Colony PCR.
 +
|}
We used 25 different colonies.
We used 25 different colonies.
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* DNA : 2µL
* DNA : 2µL
* dNTP : 0.5µL
* dNTP : 0.5µL
-
* Dream Taq : ... cf cahier !!!!!!!
+
* Dream Taq : 0.25µL
-
* H2O : ... cf cahier !!!!!
+
* H2O : 25µL
===='''2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α'''====
===='''2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α'''====
Line 191: Line 262:
{|
{|
-
| style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel3007.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
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Expected sizes :  
Expected sizes :  
-
* Bba_K1155002 : ...
+
* Bba_K1155002 : 169bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION !!!!!!!
+
We obtain fragments at the right size. We will sequence it.
|}
|}
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/July/26|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/July/31|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 00:58, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 30

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Inactivation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB

Xavier

Protocol : Ethanol precipitation

We used 30µL of DNA.

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Tranformation of BBa_K1155003 in DH5α

Abdou

Protocol : Bacterial transformation

2 - Digestion of BBa_I732017 by EcoRI/PstI

Zhou

Used quantities :

  • BBa_I732017 : 10µL
  • Buffer FD : 3µL
  • PstI FD : 1.5µL
  • EcoRI FD : 1.5µL
  • H2O : 14µL

We let the digestion 1h30 at 37°C.

3 - Digestion of BBa_I732017 by EcoRI/SpeI

Abdou

We used wrong enzymes so we will do the digestion again.

Used quantities :

  • BBa_I732017 : 10µL
  • Buffer FD : 2µL
  • SpeI FD : 1µL
  • EcoRI FD : 1µL
  • H2O : 6µL

We let the digestion 1h30 at 37°C.

4 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI

Zhou

Psgel23007.jpg
  • Well 1 : 10µL of DNA Ladder
  • Well 2 : 10µL of BBa_I732017 digested by EcoRI/SpeI+4µL of 6X loading dye
  • Well 3 : 10µL of BBa_I732017 digested by EcoRI/SpeI+4µL of 6X loading dye
  • Gel : 1%

Expected size :

  • RBS-LacZ : 3090bp

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

5 - Gel purification of digestion of BBa_I732017 by EcoRI/SpeI

Xavier

Protocol : Gel purification

We lost our fragments. We will do the digestion again.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - PCR of : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Abdou, Xavier

Used quantities :

  • Bphr2 Part I :
    • Oligo 54F : 4µL
    • Oligo 55R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA of Pseudomonas pseudoalcaligenes : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL
  • Bphr2 Part II :
    • Oligo 56F : 4µL
    • Oligo 57R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA Pseudomonas pseudoalcaligenes : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL
  • RBS-Bphr2 Part I :
    • Oligo 58F : 4µL
    • Oligo 57R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA Pseudomonas pseudoalcaligenes : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL
  • FNR Part I :
    • Oligo 59F : 4µL
    • Oligo 60R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA Escherichia coli : 2µL
    • dNTP : 4µL
    • Dream taq : 1µL
    • H2O : 146µL
  • FNR Part II :
    • Oligo 61F : 4µL
    • Oligo 62R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA Escherichia coli : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL
  • RBS-FNR Part I :
    • Oligo 63F : 4µL
    • Oligo 62R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA Escherichia coli : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL
  • PSB1C3 :
    • Prefixe 51 : 4µL
    • Suffixe 52 : 4µL
    • Buffer Dream Taq : 40µL
    • DNA : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL

PCR Program :

  • BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

PsPCRBphR23007.jpg

  • FNR Part I, FNR Part II, RBS-FNR Part I :

PsPCRFNR3007.jpg

2 - Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Psgel13007.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of PSB1C3+1µl of 6X loading dye
  • Well 3 : 5µL of PSB1C3+1µl of 6X loading dye
  • Well 4 : 5µL of BphR2 Part I+1µl of 6X loading dye
  • Well 5 : 5µL of BphR2 Part II+1µl of 6X loading dye
  • Well 6 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
  • Well 7 : 5µL of FNR Part I+1µl of 6X loading dye
  • Well 8 : 5µL of FNR Part II+1µl of 6X loading dye
  • Well 9 : 5µL of RBS-FNR Part I+1µl of 6X loading dye
  • Gel : 0.8%

Expected size

  • BphR2 Part I : 178 kb
  • BphR2 Part II : 790 kb
  • RBS-BphR2 Part I : 197 kb
  • FNR Part I : 597 kb
  • FNR Part II : 200 kb
  • RBS-FNR Part I : 615 kb
  • PSB1C3 : 2070bp

We can't see RBS-BphR2 Part I fragments at the good size. We will make the PCR again with a new PCR Program. We obtain BphR2 Part I, BphR2 Part II, FNR Part I, Fnr Part II and RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.

3 - PCR of : RBS-BphR2 Part I

Abdou, Xavier

Used quantities :

    • Oligo 54F : 4µL
    • Oligo 55R : 4µL
    • Buffer Dream Taq : 40µL
    • DNA : 2µL
    • dNTP : 4µL
    • Dream Taq : 1µL
    • H2O : 146µL

PCR Program :

PsPCRBSBphR23007.jpg

4 - Gel purification of PCR of : RBS-BphR2 Part I

Xavier

Protocol : Gel purification

We lost our fragments. We will do the PCR again.


B - PCB sensor system

Objective : obtaining Bba_K1155002

1 - Colony PCR of Bba_K1155002 in DH5α

Abdou, Xavier

The transformation was good. We will make a Colony PCR.

We used 25 different colonies.

Used quantities :

  • Oligo 43F : 0.5µL
  • Oligo 44R : 0.5µL
  • Buffer Dream Taq : 35µL
  • DNA : 2µL
  • dNTP : 0.5µL
  • Dream Taq : 0.25µL
  • H2O : 25µL

2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α

Abdou, Xavier

Psgel3007.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 to 26 : 5µL of Bba_K1155002+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Bba_K1155002 : 169bp

We obtain fragments at the right size. We will sequence it.


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