Team:Paris Saclay/Notebook/July/31

From 2013.igem.org

(Difference between revisions)
(→4 -Electrophoresis to check the ligation of NarK, NarG, NirB in PSB1C3)
Line 9: Line 9:
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''====
===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K155006'''====
-
===='''1 -Digestion of PSB1C3 by EcoRI/PstI'''====  
+
===='''1 -Digestion of pSB1C3 by EcoRI/PstI'''====  
Abdou  
Abdou  
Line 21: Line 21:
We let our digestion 1h30 at 37°C.
We let our digestion 1h30 at 37°C.
-
===='''2 - Inactivation of EcoRI/PstI used for the digestion of PSB1C3'''====  
+
===='''2 - Inactivation of EcoRI/PstI used for the digestion of pSB1C3'''====  
Abdou
Abdou
Line 27: Line 27:
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
Protocol : [[Team:Paris_Saclay/ethanol|Ethanol precipitation]]
-
===='''3 -Ligation of NarK, NarG, NirB in PSB1C3'''====  
+
===='''3 -Ligation of NarK, NarG, NirB in pSB1C3'''====  
   
   
XiaoJing
XiaoJing
Line 34: Line 34:
* Buffer : 2µL
* Buffer : 2µL
* NarK, NarG, NirB : 1µL  
* NarK, NarG, NirB : 1µL  
-
* PSB1C3 : 2µL
+
* pSB1C3 : 2µL
* Ligase : 1µL
* Ligase : 1µL
* H2O : 14µL
* H2O : 14µL
-
===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in PSB1C3'''====  
+
===='''4 -Electrophoresis to check the ligation of NarK, NarG, NirB in pSB1C3'''====  
XiaoJing
XiaoJing
Line 56: Line 56:
Expected sizes :  
Expected sizes :  
-
* NarK : 200bp
+
* Nar K : 200bp
* Nar G : 200bp
* Nar G : 200bp
* Nir B : 200bp
* Nir B : 200bp

Revision as of 16:59, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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.


Previous day Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/July/31"