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_B0010, 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'''====
 +
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
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel11607.jpg|500px]]
 +
| 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 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
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
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 :
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel21607.jpg|300px]]
 +
| 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 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
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragment at the right size. We will amplify BBa_K1155001.
 +
|}
 +
 +
* BBa_K1155002 :
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel31607.jpg|500px]]
 +
| 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 9 : 6µL DNA Ladder
 +
* Well 10 to 17 : 2µL BBa_K1155002 digested by SacII+2µl of 6X loading dye
 +
* Gel : 1.5%
 +
|}
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We didn't obtain fragments at the right size. We will do the ligation again.
 +
|}
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel41607.jpg|500px]]
 +
| 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 9 : 6µL DNA Ladder
 +
* Well 10 to 17 : 2µL BphR2 in pSB1C3 digested by SacII+2µl of 6X loading dye
 +
* Gel : 1.5%
 +
|}
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
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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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
-
|[[Team:Paris Saclay/Notebook/July/17|<big>next day</big>]]
+
|[[Team:Paris Saclay/Notebook/July/17|<big>Next day</big>]]
|}
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

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 : 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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