Team:Paris Saclay/Notebook/August/12

From 2013.igem.org

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{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 9'''=
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=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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===='''1 - Obtaining Pfnr_RBS-LacZ-Term in PSB1C3'''====
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Anaïs, Nadia, XiaoJing
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We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain  was too old ( 2001) 
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Protocol : [[Team:Paris_Saclay/Protocols/transduction|transduction]]
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{|
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| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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'''Picture: lysed cells comparison.'''
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* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
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* 50µl phage: the petri dish is clear, bacteria are lysed by phages.
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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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|}
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10µl,50µl and 100µl petri dishes are clear so phages are multiplied.
 +
 +
We let the antibiotic over night to select the right strain.
 +
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===='''2 - Obtaining RBS_lacZ_ter. '''====
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Abdou
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Clone 10,14 and 15 plamid extraction using nucleospin kit.
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Protocol : [[Team:Paris_Saclay/Protocols/hight copy plamid extraction|hight copy plamid extraction]]
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Results: concentration measured by nanodrop
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*clone 10: C=38ng/µl  260/280= 1.78
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*clone 14: C=48.5ng/µl  260/280=1.90
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*clone 15: C=52 ng/µl    260/280=1.78
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We still have to sequence plasmids in order to verify our results.
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==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
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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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Results:
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{|
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| style="width:350px;border:1px solid black;" | IMAGE
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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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 3 : 5µL of BphR2 part2+1µl of 6X loading dy
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*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
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*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
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*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
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*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
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*Gel : 0.8%
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|}
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we have no fragment so we must do again these PCRs
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{{Team:Paris_Saclay/incl_fin}}

Revision as of 15:26, 12 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

1 - Obtaining Pfnr_RBS-LacZ-Term in PSB1C3

Anaïs, Nadia, XiaoJing

We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain was too old ( 2001)

Protocol : transduction

Ps908transduction.jpg

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 - Obtaining 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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