Team:Paris Saclay/Notebook/July/31

From 2013.igem.org

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(→2 -Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI)
 
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===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''====
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''====
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===='''1 -Digestion of PSB1C3 by EcoRI/PstI'''====  
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===='''1 -Digestion of pSB1C3 by EcoRI/PstI'''====  
Abdou  
Abdou  
Used quantities :  
Used quantities :  
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* PSB1C3 : 16µL
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* pSB1C3 : 16µL
* Buffer FD : 2µL
* Buffer FD : 2µL
* EcoRI FD : 1µL
* EcoRI FD : 1µL
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We let our digestion 1h30 at 37°C.
We let our digestion 1h30 at 37°C.
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===='''2 -Denaturation of EcoRI/PstI used for the digestion of PSB1C3'''====  
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===='''2 - Inactivation of EcoRI/PstI used for the digestion of pSB1C3'''====  
Abdou
Abdou
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
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+
 
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===='''3 -Ligation of NarK, NarG, NirB in PSB1C3'''====  
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===='''3 -Ligation of NarK, NarG, NirB in pSB1C3'''====  
   
   
XiaoJing
XiaoJing
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* Buffer : 2µL
* Buffer : 2µL
* NarK, NarG, NirB : 1µL  
* NarK, NarG, NirB : 1µL  
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* PSB1C3 : 2µL
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* pSB1C3 : 2µL
* Ligase : 1µL
* Ligase : 1µL
* H2O : 14µL
* H2O : 14µL
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===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in PSB1C3'''====  
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===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in pSB1C3'''====  
XiaoJing
XiaoJing
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Expected sizes :  
Expected sizes :  
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* NarK : ...
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* Nar K : 200bp
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* Nar G : ...
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* Nar G : 200bp
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* Nir B : ...
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* Nir B : 200bp
{|
{|
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Expected sizes :  
Expected sizes :  
* RBS-LacZ : 3093 bp
* RBS-LacZ : 3093 bp
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* PSB1A2 : 2079 bp
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* pSB1A2 : 2079 bp
{|
{|

Latest revision as of 01:00, 5 October 2013

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Contents

Notebook : July 31

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006

1 -Digestion of pSB1C3 by EcoRI/PstI

Abdou

Used quantities :

  • pSB1C3 : 16µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL

We let our digestion 1h30 at 37°C.

2 - Inactivation of EcoRI/PstI used for the digestion of pSB1C3

Abdou

Protocol : Ethanol precipitation

3 -Ligation of NarK, NarG, NirB in pSB1C3

XiaoJing

Used quantities :

  • Buffer : 2µL
  • NarK, NarG, NirB : 1µL
  • pSB1C3 : 2µL
  • Ligase : 1µL
  • H2O : 14µL

4 -Electrophoresis to check the ligation of NarK, NarG, NirB in pSB1C3

XiaoJing

Psgel23107.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of NirB+1µL 6X loading dye
  • Well 3 : 5µL of NirB+1µL 6X loading dye
  • Well 4 : 5µL of NarG+1µL 6X loading dye
  • Well 5 : 5µL of NarG+1µL 6X loading dye
  • Well 6 : 5µL of NarK+1µL 6X loading dye
  • Well 7 : 5µL of NarK+1µL 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Nar K : 200bp
  • Nar G : 200bp
  • Nir B : 200bp

We obtain fragments at the right size. We will transform them in DH5α.

5 -Transformation of BBa_K1155004, BBa_K1155005, BBa_K1155006

Anaïs

Protocol : Bacterial transformation

Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI

XiaoJing

Used quantities :

  • BBa_I732017 : 41µL
  • Buffer FD : 5µL
  • EcoRI : 2µL
  • SpeI : 2µL

We let the digestion at 1h30 at 37°C.

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

XiaoJing

Psgel13107.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 et 3 : 40µL of BBa_I732017 digested by EcoRI/SpeI+8µL 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093 bp
  • pSB1A2 : 2079 bp

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

3 - Gel purification of digestion of RBS-LacZ

Xavier

Protocol : Gel purification

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


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