Team:Paris Saclay/Notebook/August/12

From 2013.igem.org

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(→1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II)
 
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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : characterize Bba_K1155000'''====
+
===='''Objective : characterize BBa_K1155000'''====
-
===='''1 - Digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBaI/PstI'''====
+
===='''1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI'''====
Anaïs, Nadia, XiaoJing
Anaïs, Nadia, XiaoJing
Line 15: Line 15:
Used quantities :
Used quantities :
-
* Bba_K1155000 :  
+
* BBa_K1155000 :  
** Buffer FD : 5µL
** Buffer FD : 5µL
** H2O : 38µL
** H2O : 38µL
Line 22: Line 22:
** PstI FD : 1µL
** PstI FD : 1µL
-
* Bba_K1155007 :  
+
* BBa_K1155007 :  
** Buffer FD : 5µL  
** Buffer FD : 5µL  
** H2O : 23µL
** H2O : 23µL
Line 29: Line 29:
** PstI FD : 1µL
** PstI FD : 1µL
-
* Bba_K1155003 :  
+
* BBa_K1155003 :  
** Buffer FD : 5µL
** Buffer FD : 5µL
** H2O : 33µL
** H2O : 33µL
Line 36: Line 36:
** PstI FD : 1µL
** PstI FD : 1µL
-
We let the digestion at 37°C during 10 minutes ?????????
+
We incubated the digestion at 37°C during 15 minutes.
-
===='''2 - Electrophoresis to check the digestion of Bba_K1155000 by SpeI/PstI, Bba_K1155007 and Bba_K1155003 by XBalI/PstI'''====  
+
===='''2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI'''====  
Nadia
Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" |File:Psdigestion12.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel11208.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
-
* Well 2 : 5µL Bba_K1155000 digested by SpeI/PstI+1µl of 6X loading dye
+
* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
-
* Well 3 : 5µL Bba_K1155007 digested by XBaI/PstI+1µl of 6X loading dye
+
* Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
-
* Well 4 : 5µL Bba_K1155003 digested by xBaI/PstI+1µl of 6X loading dye
+
* Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}
|}
Expected sizes :  
Expected sizes :  
-
* Pfnr : ...
+
* Pndh* : 111bp
* RBS_LacZ_Term : 3500 kb
* RBS_LacZ_Term : 3500 kb
-
* RBS_AmilCP_Term : ...
+
* RBS_AmilCP_Term : 824 bp
-
* PSB1C3 : ...
+
* pSB1C3 : 2070bp
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We can't see any band for Bba_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.
+
We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.
|}
|}
-
===='''3 - Digestion of Bba_K1155000 by SpeI/PstI'''====
+
===='''3 - Digestion of BBa_K1155000 by Spe I/Pst I'''====
Anaïs, Nadia
Anaïs, Nadia
Line 75: Line 75:
* PstI FD : 1µL
* PstI FD : 1µL
-
We let the digestion at 37°C during 10 minutes ???????
+
We incubate the digestion at 37°C during 15 minutes.
-
 
+
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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{|
{|
-
| style="width:350px;border:1px solid black;" |[[]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel21208.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
-
* Well 2 : 5µL RBS-BphR2 Part I+1µl of 6X loading dye
+
* Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
-
* Well 3 : 5µL BphR2 Part II+1µl of 6X loading dye
+
* Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
-
* Well 4 : 5µL FNR Part I+1µl of 6X loading dye
+
* Well 4 : 5µL FNR Part I +1µl of 6X loading dye
-
* Well 5 : 5µL FNR Part II+1µl of 6X loading dye
+
* Well 5 : 5µL FNR Part II +1µl of 6X loading dye
-
* Well 6 : 5µL RBS-FNR Part I+1µl of 6X loading dye
+
* Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
-
* Well 4 : 5µL BphR2 Part II+1µl of 6X loading dye
+
* Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
* Gel : 0.8%
* Gel : 0.8%
|}
|}
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We can't see FNR Part I, FNR Part II and BphR2 Part i fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.
+
We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.
 +
|}
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Latest revision as of 19:26, 3 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000

1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI

Anaïs, Nadia, XiaoJing

Used quantities :

  • BBa_K1155000 :
    • Buffer FD : 5µL
    • H2O : 38µL
    • DNA : 5µL
    • SpeI FD : 1µL
    • PstI FD : 1µL
  • BBa_K1155007 :
    • Buffer FD : 5µL
    • H2O : 23µL
    • DNA : 20µL
    • XBal FD : 1µL
    • PstI FD : 1µL
  • BBa_K1155003 :
    • Buffer FD : 5µL
    • H2O : 33µL
    • DNA : 10µL
    • XBal FD : 1µL
    • PstI FD : 1µL

We incubated the digestion at 37°C during 15 minutes.

2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI

Nadia

Psgel11208.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
  • Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
  • Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pndh* : 111bp
  • RBS_LacZ_Term : 3500 kb
  • RBS_AmilCP_Term : 824 bp
  • pSB1C3 : 2070bp

We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.

3 - Digestion of BBa_K1155000 by Spe I/Pst I

Anaïs, Nadia

Used quantities :

  • Buffer FD : 5µL
  • H2O : 38µL
  • DNA : 5µL
  • SpeI FD : 1µL
  • PstI FD : 1µL

We incubate the digestion at 37°C during 15 minutes.

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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Damir

Psgel21208.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
  • Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
  • Well 4 : 5µL FNR Part I +1µl of 6X loading dye
  • Well 5 : 5µL FNR Part II +1µl of 6X loading dye
  • Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
  • Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
  • Gel : 0.8%

Expected size

  • RBS-BphR2 Part I : 197 kb
  • BphR2 Part II : 790 kb
  • FNR Part I : 597 kb
  • FNR Part II : 200 kb
  • RBS-FNR Part I : 615 kb
  • BphR2 Part I : 178 kb

We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.


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