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
Line 40: Line 40:
* 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
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
 +
==='''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 91: 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;" |
-
In the first well, 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_K1155001 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 : ...
 
{|
{|
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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.  
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.  
|}
|}
 +
{| border="1" align="center"
{| border="1" align="center"

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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