Team:Paris Saclay/Notebook/August/2

From 2013.igem.org

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(→Transformation the Gibson assembly mix (August 1st))
 
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : obtaining Bba_K1155003'''====
+
===='''Objective : obtaining BBa_K1155003'''====
-
===='''1 - Extraction of Bba_K1155003 from DH5α'''====
+
===='''1 - Extraction of BBa_K1155003 from DH5α'''====
Damir, Nadia
Damir, Nadia
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DE;" |
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
Tranformation from 07/30/13 works. We will extract bra_K1155003
+
Tranformation from 07/30/13 works. We will extract plasmid BBa_K1155003.
|}
|}
-
Protocol : [[Team:Paris_Saclay/Protocols/Hight copy plamid extraction|Hight copy plamid extraction]]
+
Protocol : [[Team:Paris_Saclay/extraction|High-copy plasmid extraction]]
-
We used colony 9, 11 and 12. ????????
+
We extracted plasmid from colony 9, 11 and 12.
-
Elué dans 50µL d'H2O.
+
We eluted our extracted plasmid in 50µL H2O.
-
===='''2 - Electrophoresis to check the extraction of Bba_K1155003'''====
+
===='''2 - Electrophoresis to check the extraction of BBa_K1155003'''====
Damir, Nadia
Damir, Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel10208.jpg| 400px]]
| 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 Ladder
* Well 1 : 6µL of DNA Ladder
-
* Well 2 : 5µL of Bba_K1155003 from clone 9+1µl of 6X loading dye
+
* Well 2 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
-
* Well 3 : 5µL of Bba_K1155003 from clone 11+1µl of 6X loading dye
+
* Well 3 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
-
* Well 5 : 5µL of Bba_K1155003 from clone 12+1µl of 6X loading dye
+
* Well 5 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}  
|}  
Expected sizes :  
Expected sizes :  
-
* Bba_K1155003 : 2734 pb
+
* BBa_K1155003 : 2734 pb
Estimated concentrations :  
Estimated concentrations :  
* Clone 9 : 18ng/µL
* Clone 9 : 18ng/µL
* Clone 11 :18ng/µL
* Clone 11 :18ng/µL
-
*Clone 12 : 18ng/µL
+
* Clone 12 : 18ng/µL
{|
{|
| 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 (Clone 12 starts 5 min after the others). The extraction was good. We will digest it.
+
We obtained fragments at the right size (Clone 12 starts 5 min after the others). The extraction was good. We will digest it.
|}
|}
-
===='''Objective : obtaining Bba_K1155007'''====
 
-
===='''1 - Digestion of Bba_I732017 by EcoRI/SpeI'''====  
+
===='''Objective : obtaining BBa_K1155007'''====
 +
 
 +
===='''1 - Digestion of BBa_I732017 by EcoRI/SpeI to check sizes of fragments'''====  
Damir, Nadia
Damir, Nadia
Used quantities :  
Used quantities :  
-
* Bba_I732017 : 41µL  
+
* BBa_I732017 : 41µL  
* Buffer FD : 5µL
* Buffer FD : 5µL
* EcoRI : 2µL
* EcoRI : 2µL
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We let the digestion at 1h30 at 37°C.
We let the digestion at 1h30 at 37°C.
-
===='''2 -Electrophoresis to check the digestion of Bba_I732017 by EcoRI/SpeI'''====  
+
===='''2 -Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI'''====  
Damir, Nadia
Damir, Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel20208.jpg| 400px]]
| 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 Ladder
* Well 1 : 6µL of DNA Ladder
-
* Well 2 et 3 : 5µL of Bba_I732017 digested by EcoRI/SpeI+1µL 6X loading dye
+
* Well 2 et 3 : 5µL of BBa_I732017 digested by EcoRI/SpeI + 1µL 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}
|}
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Expected sizes :  
Expected sizes :  
* RBS-LacZ : 3093 bp
* RBS-LacZ : 3093 bp
-
* PSB1A2 : 2079 bp
+
* pSB1A2 : 2079 bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We don't obtain fragments at the right size. We will digest Bba_KI732017 again.
+
We didn't obtain fragments at the right size. We will digest BBa_KI732017 again.
|}
|}
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===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
 +
XiaoJing
-
===='''1 - Tranformation of BphR2, FNR and RBS-FNR in DH5α'''====
+
====''' Transformation the Gibson assembly mix (August 1st)'''====
-
XiaoJing
+
* plasmid pSB1C3 containing BphR2
 +
* plasmid pSB1C3 containing FNR
 +
* plasmid pSB1C3 contaning RBS-FNR in competent cell DH5α
-
Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]]
+
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/1|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/5|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 21:15, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 2

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Extraction of BBa_K1155003 from DH5α

Damir, Nadia

Tranformation from 07/30/13 works. We will extract plasmid BBa_K1155003.

Protocol : High-copy plasmid extraction

We extracted plasmid from colony 9, 11 and 12. We eluted our extracted plasmid in 50µL H2O.

2 - Electrophoresis to check the extraction of BBa_K1155003

Damir, Nadia

Psgel10208.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
  • Well 3 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
  • Well 5 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • BBa_K1155003 : 2734 pb

Estimated concentrations :

  • Clone 9 : 18ng/µL
  • Clone 11 :18ng/µL
  • Clone 12 : 18ng/µL

We obtained fragments at the right size (Clone 12 starts 5 min after the others). The extraction was good. We will digest it.


Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI to check sizes of fragments

Damir, Nadia

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

Damir, Nadia

Psgel20208.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 et 3 : 5µL of BBa_I732017 digested by EcoRI/SpeI + 1µL 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • RBS-LacZ : 3093 bp
  • pSB1A2 : 2079 bp

We didn't obtain fragments at the right size. We will digest BBa_KI732017 again.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

XiaoJing

Transformation the Gibson assembly mix (August 1st)

  • plasmid pSB1C3 containing BphR2
  • plasmid pSB1C3 containing FNR
  • plasmid pSB1C3 contaning RBS-FNR in competent cell DH5α

Protocol : Bacterial transformation


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