Team:Paris Saclay/Notebook/August/14

From 2013.igem.org

(Difference between revisions)
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**ADN : 20µL
**ADN : 20µL
**H20 : 3µL  
**H20 : 3µL  
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===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
 
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=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
 
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Anaïs
 
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*Colony counting :
 
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**Low concentration petri dish : 47 colonies
 
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**High concentration petri dish : 145 colonies
 
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*Picking of 25 colonies
 
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*Preparation of 700µL of Master mix
 
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**H<sub>2</sub>O : 590µL
 
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**dNTP : 28µL
 
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**VF2 primer : 3.5µL
 
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**VR primer : 3.5µL
 
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**DreamTaq buffer 10x : 70µL
 
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**DreamTaq enzyme : 5µL
 
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Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
 
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PCR Program :
 
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[[File:PsPcr808.jpg|400px]]
 
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=====2 - Gel electrophoresis of the colony PCR products=====
 
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Anaïs, Damir
 
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{|
 
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| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
 
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| style="width:350px;border:1px solid black;vertical-align:top;" |
 
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*6µL DNA Ladder
 
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*10µL sample per well
 
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*Gel : 0.8%
 
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|}
 
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Expected size : 3583bp
 
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Colonies 10, 14, 15 exhibit plasmids with the right length.
 
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====3 - PCR product (made the 08/01/2013) purification====
 
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Damir
 
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available quantity:
 
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* FNR Part1 : 10 µl
 
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* FNR Part2 : 19 µl
 
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* RBS FNR Part1 :16.1µl
 
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* RBS BphR2 Part1 : 28µl
 
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* BphR2 Part1 : 16.4 µl
 
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* BphR2 Part2 : 18.9 µl
 
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Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
 
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<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
 
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{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 20:40, 18 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 14

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the NarK or NarG or NirB _RBS-LacZ-term in PSB1C3 and in PSB3K3

1 - Gel electrophoresis to check the digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by EcoRI and SpeI

Anaïs, Nadia

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_K1155006 digested by EcoRI/SpeI
  • Well 3 : 5µL of BBa_K1155005 digested by EcoRI/SpeI
  • Well 4 : 5µL of BBa_K1155004 digested by EcoRI/SpeI
  • Gel : 1%

Expected sizes :

  • PSB1C33 : 2070kb
  • NarK, NarG, NirB : 200kb 
We obtained fragments of the right size but in very few quantity. We do it again but this time we will use more quantity of enzymes AND ADN ?!
2 - Digestion of Bba_K1155004,Bba_K1155005, Bba_K1155006 by SpeI and EcoRI/SpeI

Anaïs, Nadia

Protocol : Digestion

  • SpeI :
    • Buffer : 2µL
    • SpeI : 2µL
    • ADN : 15µL
    • H20 : 1µL
  • EcoRI/SpeI :
    • Buffer : 3µL
    • SpeI : 2µL
    • EcoRI : 2µL
    • ADN : 20µL
    • H20 : 3µL


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