Team:Paris Saclay/Notebook/July/1

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(2 - Ligation of pSB1C3 and Pndh*)
(1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI)
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** H2O : 5.5µL
** H2O : 5.5µL
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We let the digestion at 37°C during 3 hours.
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We incubate the digestion at 37°C during 3 hours.
===='''2 - Ligation of pSB1C3 and Pndh*'''====
===='''2 - Ligation of pSB1C3 and Pndh*'''====

Revision as of 00:45, 5 October 2013

Contents

Notebook : July 1

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155000

1 - Digestion of pSB1C3 plasmid and PCR products : Pndh* by EcoRI/ PstI

Zhou

We did a PCR amplification. The products were good. After we made digestion by EcoRI/PstI.

Used quantities :

  • pSB1C3 :
    • Plasmid : 4µL
    • EcoRI FD : 0.5µL
    • PstI FD : 0.5µL
    • Buffer FD : 0.8µL
    • H2O : 2.2µL
  • Pndh* :
    • Pndh* : 20µL
    • EcoRI FD : 0.75µL
    • PstI FD : 0.75µL
    • Buffer FD : 3µL
    • H2O : 5.5µL

We incubate the digestion at 37°C during 3 hours.

2 - Ligation of pSB1C3 and Pndh*

Sheng, Zhou

Used quantities :

  • Mix A : we mix our digestion mixes :
    • Digestion mix of pSB1C3 : 4µL
    • Digestion mix of Pndh* : 30µL
    • Buffer ligation : 2µL
    • H2O : 14µL
  • Then, we denature EcoRI/SpeI used for the digestion by ethanol precipitation.

Protocol : Ethanol precipitation

We used 50µL of DNA. At the end, we dilute our DNA in 20µL of H2O.

  • Finally we did the ligation mix :
    • Buffer ligation : 2µL
    • Ligase : 1µL
    • DNA : 2µL
    • H2O : 15µL

We let the ligation 1h30.

B - PCB sensor system

Objective : obtaining BphR2 protein

1 - Design of oligos for amplification of BphR2 gene of Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans

Abdou, Sheng, Zhou

We used software gene manager to find the correct oligopeptides for amplification of BphR2 genes in Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans.


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