Team:Paris Saclay/Notebook/August/12

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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'''===
-
'''Obtaining Pfnr_RBS-LacZ-Term in PSB1C3'''
+
===='''Objective : characterize BBa_K1155000'''====
-
===='''1 - Digestion of Bba_K1155000, Bba_K1155007 and Bba_K1155003'''====
+
===='''1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI'''====
Anaïs, Nadia, XiaoJing
Anaïs, Nadia, XiaoJing
-
Protocol : [[Team:Paris_Saclay/Protocols/digestion|digestion]]
+
Used quantities :
-
Used quantities :
+
* BBa_K1155000 :  
-
* Pfnr in PSB1C3 (Bba_K1155000) :  
+
** Buffer FD : 5µL
-
** Buffer : 5µL
+
** H2O : 38µL
** H2O : 38µL
-
** Pfnr : 5µL
+
** DNA : 5µL
-
** SpeI : 1µL
+
** SpeI FD : 1µL
-
** PstI : 1µL
+
** PstI FD : 1µL
-
* RBS-LacZ-Term in PSB1C3 (Bba_K1155007) :  
+
* BBa_K1155007 :  
-
** Buffer : 5µL  
+
** Buffer FD : 5µL  
** H2O : 23µL
** H2O : 23µL
-
** RBS-LacZ-Term : 20µL  
+
** DNA : 20µL  
-
** XBal : 1µL
+
** XBal FD : 1µL
-
** PstI : 1µL
+
** PstI FD : 1µL
-
* RBS-AmilCP-Term in PSB1C3 (Bba_K1155003) :  
+
* BBa_K1155003 :  
-
** Buffer : 5µL
+
** Buffer FD : 5µL
** H2O : 33µL
** H2O : 33µL
-
** RBS-AmilCP-Term : 10µL  
+
** DNA : 10µL  
-
** XBal : 1µL
+
** XBal FD : 1µL
-
** PstI : 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
{|
{|
-
| 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 of digested Bba_K1155000+1µl of 6X loading dy
+
* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
-
*Well 3 : 5µL of digested Bba_K1155007+1µl of 6X loading dy
+
* Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
-
*Well 4 : 5µL of digested Bba_K1155003+1µl of 6X loading dy
+
* Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
-
*Gel : 0.8%
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* Gel : 0.8%
|}
|}
Expected sizes :  
Expected sizes :  
-
*RBS_LacZ_Term : 3500 kb
+
* Pndh* : 111bp
-
*RBS_AmilCP_Term :  
+
* RBS_LacZ_Term : 3500 kb
-
*PSB1C3 :  
+
* RBS_AmilCP_Term : 824 bp
 +
* pSB1C3 : 2070bp
-
===='''2 - ObtaµLining RBS_lacZ_ter. '''====
+
{|
-
Abdou
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| 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.
 +
|}
-
Clone 10,14 and 15 plamid extraction using nucleospin kit.
+
===='''3 - Digestion of BBa_K1155000 by Spe I/Pst I'''====
-
Protocol : [[Team:Paris_Saclay/Protocols/hight copy plamid extraction|hight copy plamid extraction]]
+
Anaïs, Nadia
 +
 +
Used quantities :
-
Results: concentration measured by nanodrop
+
* Buffer FD : 5µL
-
*clone 10: C=38ng/µl  260/280= 1.78
+
* H2O : 38µL
-
*clone 14: C=48.5ng/µl  260/280=1.90
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* DNA : 5µL
-
*clone 15: C=52 ng/µl    260/280=1.78
+
* SpeI FD : 1µL
-
We still have to sequence plasmids in order to verify our results.
+
* PstI FD : 1µL
-
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
+
We incubate the digestion at 37°C during 15 minutes.
-
===='''1 - Gibson assembly.'''====
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-
August 1st PCR purification to be sure about the experiment.
+
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BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.
+
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Protocol : [[Team:Paris_Saclay/Protocols/PCR_clean_up|PCR_clean_up]]
+
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-
Results:
+
===='''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
{|
{|
-
| style="width:350px;border:1px solid black;" | IMAGE
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| 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;" |
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*Well 1 : 6µL DNA Ladder
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* Well 1 : 6µL DNA Ladder
-
*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
+
* Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
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*Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
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* Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
-
*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
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* Well 4 : 5µL FNR Part I +1µl of 6X loading dye
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*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
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* Well 5 : 5µL FNR Part II +1µl of 6X loading dye
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*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
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* Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
-
*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
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* Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
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*Gel : 0.8%
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* 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
 +
 
 +
{|
 +
| 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.
 +
|}
 +
 
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{| border="1" align="center"
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 +
 
 +
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|}
|}
-
we have no fragment so we must do again these PCRs
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 19:26, 3 October 2013

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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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