Team:Paris Saclay/Notebook/July/16

From 2013.igem.org

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(→1 - Electrophoresis of the digestion of BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3)
 
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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 - Extraction of Bba_B0015, Bba_B0017, Bba_I732019 from DH5α'''====
+
===='''1 - Extraction of BBa_B0015, BBa_B0017, BBa_I732019 from DH5α'''====
Zhou
Zhou
Line 15: Line 15:
Protocol : [[Team:Paris_Saclay/extraction|High copy plamid extraction]]
Protocol : [[Team:Paris_Saclay/extraction|High copy plamid extraction]]
-
===='''2 - Digestion of Bba_B0015, Bba_B0017, Bba_I732019 by EcoRI/PstI'''====
+
===='''2 - Digestion of BBa_B0015, BBa_B0017, BBa_I732019 by EcoRI/PstI'''====
Anaïs
Anaïs
Line 29: Line 29:
===='''Objective : obtaining biobricks in PSB3K3'''====
===='''Objective : obtaining biobricks in PSB3K3'''====
-
===='''1 - Digestion of PSB3K3 by EcoRI/PstI'''====
+
===='''1 - Digestion of pSB3K3 by EcoRI/PstI'''====
Anaïs
Anaïs
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* H2O : 11µL
* H2O : 11µL
-
We let our digestion 1h30 at 37°C.
+
We let our digestion 1h30 at 37°C.p
-
 
+
===='''2 - Electrophoresis of the digestion of pSB3K3 by EcoRI/PstI'''====
-
===='''2 - Electrophoresis of the digestion of PSB3K3 by EcoRI/PstI'''====
+
Sheng
Sheng
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel11607.jpg|500px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
+
* Well 1 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
-
* Well 2 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
+
* Well 2 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
* Well 3 : 6µL of DNA ladder
* Well 3 : 6µL of DNA ladder
-
* Well 4 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
+
* Well 4 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
-
* Well 5 : 5µL of PSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
+
* Well 5 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of  6X loading dye
* Gel : 1%
* Gel : 1%
|}
|}
Expected sizes :  
Expected sizes :  
-
* PSB3K3 : 2750bp
+
* pSB3K3 : 2750bp
{|
{|
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|}
|}
-
===='''3 - Gel purification of electrophoresis of the digestion of PSB3K3 by EcoRI/PstI'''====
+
===='''3 - Gel purification of electrophoresis of the digestion of pSB3K3 by EcoRI/PstI'''====
Abdou
Abdou
Line 74: Line 73:
==='''B - PCB sensor system'''===
==='''B - PCB sensor system'''===
-
===='''Objective : obtaining Bba_K1155001, Bba_K1155002, BphR2 in PSB1C3'''====
+
===='''Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3'''====
-
===='''1 - Electrophoresis of the digestion of Bba_K1155001, Bba_K1155002, BphR2 in PSB1C3'''====
+
===='''1 - Electrophoresis of the digestion of BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3'''====
Anaïs, Zhou
Anaïs, Zhou
-
* Bba_K1155001 :  
+
* BBa_K1155001 :  
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel21607.jpg|300px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 to 5 : 2µL Bba_K1155001 digested by EcoRI/PstI+2µl of 6X loading dye
+
* Well 1 to 5 : 2µL BBa_K1155001 digested by EcoRI/PstI+2µl of 6X loading dye
-
* Well 6 and 7 : 2µL Bba_K1155001 digested by SacII+2µl of 6X loading dye
+
* Well 6 and 7 : 2µL BBa_K1155001 digested by SacII+2µl of 6X loading dye
* Well 8 : 6µL DNA Ladder
* Well 8 : 6µL DNA Ladder
* Gel : 1.2%
* Gel : 1.2%
Line 92: Line 91:
Expected size :  
Expected size :  
-
* Bba_K1155001 digested by EcoRI/PstI : 2037bp + 333bp
+
* BBa_K1155001 digested by EcoRI/PstI : 2037bp + 333bp
-
* Bba_K1155001 digested by SacII : 2370bp
+
* BBa_K1155001 digested by SacII : 2370bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain fragment at the right size. We will amplify Bba_K1155001.
+
We obtain fragment at the right size. We will amplify BBa_K1155001.
|}
|}
-
* Bba_K1155002 :  
+
* BBa_K1155002 :  
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel31607.jpg|500px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 to 8 : 2µL Bba_K1155002 digested by EcoRI/PstI+2µl of 6X loading dye
+
* Well 1 to 8 : 2µL BBa_K1155002 digested by EcoRI/PstI+2µl of 6X loading dye
* Well 9 : 6µL DNA Ladder
* Well 9 : 6µL DNA Ladder
-
* Well 10 to 17 : 2µL Bba_K1155002 digested by SacII+2µl of 6X loading dye
+
* Well 10 to 17 : 2µL BBa_K1155002 digested by SacII+2µl of 6X loading dye
* Gel : 1.5%
* Gel : 1.5%
|}
|}
-
 
-
Expected size :
 
-
* Bba_K1155002 digested by EcoRI/PstI : ...
 
-
* Bba_K1155002 digested by SacII : ...
 
{|
{|
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We didn't obtain fragments at the right size. We will do the ligation again.  
We didn't obtain fragments at the right size. We will do the ligation again.  
|}
|}
-
 
-
* BphR2 in PSB1C3 :
 
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel41607.jpg|500px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
* Well 1 to 8 : 2µL BphR2 in PSB1C3 digested by EcoRI/PstI+2µl of 6X loading dye
+
* Well 1 to 8 : 2µL BphR2 in pSB1C3 digested by EcoRI/PstI+2µl of 6X loading dye
* Well 9 : 6µL DNA Ladder
* Well 9 : 6µL DNA Ladder
-
* Well 10 to 17 : 2µL BphR2 in PSB1C3 digested by SacII+2µl of 6X loading dye
+
* Well 10 to 17 : 2µL BphR2 in pSB1C3 digested by SacII+2µl of 6X loading dye
* Gel : 1.5%
* Gel : 1.5%
|}
|}
-
 
-
Expected size :
 
-
* BphR2 digested by EcoRI/PstI : ...
 
-
* BphR2 digested by SacII : ...
 
{|
{|

Latest revision as of 00:02, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 16

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003, BBa_K1155007

1 - Extraction of BBa_B0015, BBa_B0017, BBa_I732019 from DH5α

Zhou

Protocol : High copy plamid extraction

2 - Digestion of BBa_B0015, BBa_B0017, BBa_I732019 by EcoRI/PstI

Anaïs

  • DNA : 5µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL
  • H2O : 21µL

We let our digestion 1h30 at 37°C.

Objective : obtaining biobricks in PSB3K3

1 - Digestion of pSB3K3 by EcoRI/PstI

Anaïs

Used quantities :

  • DNA : 5µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL
  • H2O : 11µL

We let our digestion 1h30 at 37°C.p

2 - Electrophoresis of the digestion of pSB3K3 by EcoRI/PstI

Sheng

Psgel11607.jpg
  • Well 1 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 2 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 3 : 6µL of DNA ladder
  • Well 4 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 5 : 5µL of pSB3K3 digested by EcoRI/PstI+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB3K3 : 2750bp

We obtain fragments at the right size.

3 - Gel purification of electrophoresis of the digestion of pSB3K3 by EcoRI/PstI

Abdou

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


B - PCB sensor system

Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3

1 - Electrophoresis of the digestion of BBa_K1155001, BBa_K1155002, BphR2 in pSB1C3

Anaïs, Zhou

  • BBa_K1155001 :
Psgel21607.jpg
  • Well 1 to 5 : 2µL BBa_K1155001 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 6 and 7 : 2µL BBa_K1155001 digested by SacII+2µl of 6X loading dye
  • Well 8 : 6µL DNA Ladder
  • Gel : 1.2%

Expected size :

  • BBa_K1155001 digested by EcoRI/PstI : 2037bp + 333bp
  • BBa_K1155001 digested by SacII : 2370bp

We obtain fragment at the right size. We will amplify BBa_K1155001.

  • BBa_K1155002 :
Psgel31607.jpg
  • Well 1 to 8 : 2µL BBa_K1155002 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 9 : 6µL DNA Ladder
  • Well 10 to 17 : 2µL BBa_K1155002 digested by SacII+2µl of 6X loading dye
  • Gel : 1.5%

We didn't obtain fragments at the right size. We will do the ligation again.

Psgel41607.jpg
  • Well 1 to 8 : 2µL BphR2 in pSB1C3 digested by EcoRI/PstI+2µl of 6X loading dye
  • Well 9 : 6µL DNA Ladder
  • Well 10 to 17 : 2µL BphR2 in pSB1C3 digested by SacII+2µl of 6X loading dye
  • Gel : 1.5%

We didn't obtain fragments at the right size. After reading the sequence again, we find an digestion site in the middle of our biobrick. We will do a Gibson assembly to modify this site.


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