Team:Paris Saclay/Notebook/July/1

From 2013.igem.org

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='''Notebook : August 23'''=
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=='''Lab work'''==
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==='''A - Aerobic/Anaerobic regulation system'''===
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===='''Objective : obtaining Bba_K1155000'''====
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===='''1 - Digestion of PSB1C3 plasmid and PCR products : Pfnr by EcoRI/ PstI'''====
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Zhou
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Used quantities :
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* PSB1C3 :
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** Plasmid : 4µL
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** EcoRI FD : 0.5µL
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** PstI FD : 0.5µL
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** Buffer FD : 0.8µL
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** H2O : 2.2µL
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* Pfnr :
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** Pfnr : 20µL
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** EcoRI FD : 0.75µL
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** PstI FD : 0.75µL
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** Buffer FD : 3µL
 +
** H2O : 5.5µL
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Revision as of 12:40, 22 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 1

summary

For regulator system:

  • started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoRI and PstI.
  • Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which was created by iGEM UCL team in 2009

For PCBs sensor system:

  • Designed oligonucleotides for the gene coding for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group


lab work


  • A.aero/anaerobic regulation system
    • 1.BioBrick promotor fnr(repressor) in plasmid PSB1C3


Digestion for fnr and PSB1C3


2 enzymes EcoR I and PST I can be used in one common buffer: orange buffer (10X).
For PCR products:
PCR products 20µl
EcoR I 0.75µl
PST I 0.75µl
H2O 5.5µl
buffer 3µl
total 30µl


For plasmid PSB1C3:
Plasmid 4µl
EcoR I 0.5µl
PST I 0.5µl
H2O 2.2µl
buffer 0.8µl
total 8µl


Ligation
After 3h of digestion, we mixed the digestion products:
PCR product 30µl
PSB1C3 4µl
Ligation buffer 2µl
H2O 14µl

Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
mixture control
2µl ligation buffer 2µl ligation buffer
1µl ligase T4 1µl ligase T4+2µl PSB1C3
17µl H2O 15µl H2O


The incubation was during 1H30.


  • B.PCBs sensor system
    • 1.BioBrick BphR2(regulator) in plasmid PSB1C3


Using software gene manager to find the oligopeptide for amplification of BphR2.


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Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 23

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining Bba_K1155000

1 - Digestion of PSB1C3 plasmid and PCR products : Pfnr by EcoRI/ PstI

Zhou

Used quantities :

  • PSB1C3 :
    • Plasmid : 4µL
    • EcoRI FD : 0.5µL
    • PstI FD : 0.5µL
    • Buffer FD : 0.8µL
    • H2O : 2.2µL
  • Pfnr :
    • Pfnr : 20µL
    • EcoRI FD : 0.75µL
    • PstI FD : 0.75µL
    • Buffer FD : 3µL
    • H2O : 5.5µL

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