Team:Paris Saclay/Notebook/July/23

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : obtaining Bba_K1155003, Bba_K1155007'''====
+
===='''Objective : obtaining BBa_K1155003, BBa_K1155007'''====
-
===='''1 - Electrophoresis of the digestion of Bba_I732017 by EcoRI/SpeI,  Bba_K592009 by SpeI/PstI, Bba_B0015, Bba_B0017 by XbaI/PstI '''====
+
===='''1 - Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI,  BBa_K592009 by SpeI/PstI, BBa_B0015, BBa_B0017 by XbaI/PstI '''====
Abdou, Anaïs, Xavier, Zhou
Abdou, Anaïs, Xavier, Zhou
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgellast2307.jpg|200px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 to 3: 30µL of Bba_I732017 digested by EcoRI/SpeI+6µL of 6X loading dye
+
* Well 1 to 3: 30µL of BBa_I732017 digested by EcoRI/SpeI+6µL of 6X loading dye
* Well 4 : 6µL of DNA 6X loading dye
* Well 4 : 6µL of DNA 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
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{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel22307.jpg|200px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 : 5µL of Bba_B0015 digested by XbaI/PstI+1µL of 6X loading dye
+
* Well 1 : 5µL of BBa_B0015 digested by XbaI/PstI+1µL of 6X loading dye
-
* Well 2 : 5µL of Bba_B0017 digested by XbaI/PstI+1µL of 6X loading dye
+
* Well 2 : 5µL of BBa_B0017 digested by XbaI/PstI+1µL of 6X loading dye
-
* Well 3 : 5µL of Bba_K592009 digested SpeI/PstI+1µL of 6X loading dye
+
* Well 3 : 5µL of BBa_K592009 digested SpeI/PstI+1µL of 6X loading dye
* Well 4 : 6µL of DNA 6X loading dye
* Well 4 : 6µL of DNA 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
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Expected sizes :  
Expected sizes :  
-
* RBS-LacZ :  
+
* RBS-LacZ : 3093bp
-
* Amil CP :
+
* Term : 150bp
-
* Term :  
+
* pSB3A2 : 2079bp
-
* PSB3A1 :  
+
{|
{|
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|}
|}
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===='''2 - Gel purification of digestion products : RBS-LacZ'''====
+
===='''2 -Gel purification of digestion products : RBS-LacZ'''====
Xavier
Xavier
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{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel32307.jpg|200px]]
| 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 6X loading dye
* Well 1 : 6µL of DNA 6X loading dye
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Expected sizes :  
Expected sizes :  
-
* RBS-LacZ :  
+
* RBS-LacZ : 3093bp
{|
{|
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|}
|}
-
===='''4 - Digestion of Bba_I732017 by EcoRI/PstI'''====
+
===='''4 - Digestion of BBa_I732017 by EcoRI/PstI'''====
Anaïs
Anaïs
Used quantities :  
Used quantities :  
-
* Bba_I732017 : 10µL  
+
* BBa_I732017 : 10µL  
* Buffer FD : 3µL  
* Buffer FD : 3µL  
* EcoRI FD : 1.5µL  
* EcoRI FD : 1.5µL  
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* H2O : 14µL  
* H2O : 14µL  
-
===='''5 - Electrophoresis to check the gel digestion of Bba_I732017 by EcoRI/SpeI'''====
+
===='''5 - Electrophoresis to check the gel digestion of BBa_I732017 by EcoRI/SpeI'''====
Zhou
Zhou
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel42307.jpg|200px]]
| 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 6X loading dye
* Well 1 : 6µL of DNA 6X loading dye
-
* Well 2 to 4 : 5µL of Bba_I732017 digested by EcoRI/SpeI+1µL of 6X loading dye
+
* Well 2 to 4 : 5µL of BBa_I732017 digested by EcoRI/SpeI+1µL of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}
|}
Expected sizes :  
Expected sizes :  
-
* RBS-LacZ :  
+
* RBS-LacZ : 3093bp
{|
{|
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|}
|}
-
===='''6 - Digestion of Bba_K592009 by SpeI/PstI'''====
+
===='''6 - Digestion of BBa_K592009 by SpeI/PstI'''====
Anaïs
Anaïs
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* H2O : 8µL
* H2O : 8µL
-
===='''7 - Electrophoresis to check the digestion of Bba_K592009 by SpeI/PstI'''====  
+
===='''7 - Electrophoresis to check the digestion of BBa_K592009 by SpeI/PstI'''====  
Zhou
Zhou
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel12307.jpg|200px]]
| 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 6X loading dye
* Well 1 : 6µL of DNA 6X loading dye
-
* Well 2 to 4 : 5µL of Bba_K592009 digested by SpeI/PstI+1µL of 6X loading dye
+
* Well 2 to 4 : 5µL of BBa_K592009 digested by SpeI/PstI+1µL of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}
|}
-
 
-
Expected sizes :
 
-
* Amil CP :
 
{|
{|
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|}
|}
-
===='''8 - Liquid culture of Bba_I732017 and Bba_K592009 in DH5α'''====
+
===='''8 - Liquid culture of BBa_I732017 and BBa_K592009 in DH5α'''====
Abdou, Xavier
Abdou, Xavier

Latest revision as of 00:15, 5 October 2013

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Contents

Notebook : July 23

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI, BBa_K592009 by SpeI/PstI, BBa_B0015, BBa_B0017 by XbaI/PstI

Abdou, Anaïs, Xavier, Zhou

Psgellast2307.jpg
  • Well 1 to 3: 30µL of BBa_I732017 digested by EcoRI/SpeI+6µL of 6X loading dye
  • Well 4 : 6µL of DNA 6X loading dye
  • Gel : 0.8%
Psgel22307.jpg
  • Well 1 : 5µL of BBa_B0015 digested by XbaI/PstI+1µL of 6X loading dye
  • Well 2 : 5µL of BBa_B0017 digested by XbaI/PstI+1µL of 6X loading dye
  • Well 3 : 5µL of BBa_K592009 digested SpeI/PstI+1µL of 6X loading dye
  • Well 4 : 6µL of DNA 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp
  • Term : 150bp
  • pSB3A2 : 2079bp

We obtain a RBS-lacZ and Term fragments at the right size. The digestions were good and we will purify them.

2 -Gel purification of digestion products : RBS-LacZ

Xavier

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

3 - Electrophoresis to check the gel purification of digestion products : RBS-LacZ

Abdou

Psgel32307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2: 5µL of RBS-LacZ+1µL of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp

We can't see anything. The purification wasn't good. We will do the digestion again.

4 - Digestion of BBa_I732017 by EcoRI/PstI

Anaïs

Used quantities :

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

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

Zhou

Psgel42307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2 to 4 : 5µL of BBa_I732017 digested by EcoRI/SpeI+1µL of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093bp

We can't see anything. The digestion wasn't good, we will do it the again.

6 - Digestion of BBa_K592009 by SpeI/PstI

Anaïs

Used quantities :

  • Buffer FD : 3µL
  • DNA : 15µL
  • SpeI FD : 2µL
  • PstI : 2µL
  • H2O : 8µL

7 - Electrophoresis to check the digestion of BBa_K592009 by SpeI/PstI

Zhou

Psgel12307.jpg
  • Well 1 : 6µL of DNA 6X loading dye
  • Well 2 to 4 : 5µL of BBa_K592009 digested by SpeI/PstI+1µL of 6X loading dye
  • Gel : 0.8%

We can't see anything. The purification wasn't good. We will do cultures again.

8 - Liquid culture of BBa_I732017 and BBa_K592009 in DH5α

Abdou, Xavier

We let our preparations at 37°C, 150rpm with 5mL of LB and 5mL of chloramphenicol antibiotic.

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