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 :
 +
 
 +
* 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
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.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;" |
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'''Picture: lysed cells comparison.'''
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* Well 1 : 6µL DNA Ladder
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* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
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* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
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* 50µl phage: the petri dish is clear, bacteria are lysed by phages.
+
* Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
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*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.  
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* Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
 +
* Gel : 0.8%
|}
|}
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10µl,50µl and 100µl petri dishes are clear so phages are multiplied.
+
Expected sizes :
-
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* Pndh* : 111bp
-
We let the antibiotic over night to select the right strain.
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* RBS_LacZ_Term : 3500 kb
 +
* RBS_AmilCP_Term : 824 bp
 +
* pSB1C3 : 2070bp
-
===='''2 - Obtaining RBS_lacZ_ter. '''====
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{|
-
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.
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===='''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
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* H2O : 38µL
-
*clone 14: C=48.5ng/µl  260/280=1.90
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* DNA : 5µL
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*clone 15: C=52 ng/µl    260/280=1.78
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* SpeI FD : 1µL
-
We still have to sequence plasmids in order to verify our results.
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* PstI FD : 1µL
-
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
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We incubate the digestion at 37°C during 15 minutes.
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===='''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]]
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==='''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;" |
-
*Well 1 : 6µL DNA Ladder
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* Well 1 : 6µL DNA Ladder
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*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
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* 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
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*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
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*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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