Team:Paris Saclay/Notebook/August/12

From 2013.igem.org

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Protocol : [[Team:Paris_Saclay/Protocols/digestion|digestion]]
Protocol : [[Team:Paris_Saclay/Protocols/digestion|digestion]]
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{|
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Used quantities :
 +
* Bba_K1155000 :
 +
* Buffer : 5µL
 +
* H2O : 38µL
 +
* Pfnr : 5µL
 +
* SpeI : 1µL
 +
* PstI : 1µL
 +
 
 +
* Bba_K1155007 :
 +
* Buffer : 5µL
 +
* H2O : 23µL
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* RBS-LacZ-Term : 20µL
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* XBal : 1µL
 +
* PstI : 1µL
 +
 
 +
* Bba_K1155003 :
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* Buffer : 5µL
 +
* H2O : 33µL
 +
* RBS-AmilCP-Term : 10µL
 +
* XBal : 1µL
 +
* PstI : 1µL
 +
 
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
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We let the antibiotic over night to select the right strain.  
We let the antibiotic over night to select the right strain.  
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===='''2 - Obtaining RBS_lacZ_ter. '''====
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===='''2 - ObtaµLining RBS_lacZ_ter. '''====
Abdou
Abdou

Revision as of 15:47, 12 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining Pfnr_RBS-LacZ-Term in PSB1C3

1 - Digestion of Bba_K1155000, Bba_K1155007 and Bba_K1155003

Anaïs, Nadia, XiaoJing

Protocol : digestion

Used quantities :

  • Bba_K1155000 : 
* Buffer : 5µL
* H2O : 38µL
* Pfnr : 5µL
* SpeI : 1µL
* PstI : 1µL
  • Bba_K1155007 : 
* Buffer : 5µL 
* H2O : 23µL
* RBS-LacZ-Term : 20µL 
* XBal : 1µL
* PstI : 1µL
  • Bba_K1155003 : 
* Buffer : 5µL
* H2O : 33µL
* RBS-AmilCP-Term : 10µL 
* XBal : 1µL
* PstI : 1µL

| style="width:350px;border:1px solid black;" | Ps908transduction.jpg | style="width:350px;border:1px solid black;vertical-align:top;" | Picture: lysed cells comparison.

  • 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
  • 50µl phage: the petri dish is clear, bacteria are lysed by phages.
  • 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.

|}

10µl,50µl and 100µl petri dishes are clear so phages are multiplied.

We let the antibiotic over night to select the right strain.

2 - ObtaµLining RBS_lacZ_ter.

Abdou

Clone 10,14 and 15 plamid extraction using nucleospin kit.

Protocol : hight copy plamid extraction

Results: concentration measured by nanodrop

  • clone 10: C=38ng/µl 260/280= 1.78
  • clone 14: C=48.5ng/µl 260/280=1.90
  • clone 15: C=52 ng/µl 260/280=1.78

We still have to sequence plasmids in order to verify our results.

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

1 - Gibson assembly.

August 1st PCR purification to be sure about the experiment. BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.

Protocol : PCR_clean_up

Results:

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
  • Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
  • Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
  • Well 5 : 5µL of FNR part1+1µl of 6X loading dy
  • Well 6 : 5µL of FRN part2+1µl of 6X loading dy
  • Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
  • Gel : 0.8%

we have no fragment so we must do again these PCRs

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