Team:Paris Saclay/Notebook/July/1

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(Difference between revisions)
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** Buffer FD : 3µL
** Buffer FD : 3µL
** H2O : 5.5µL
** H2O : 5.5µL
 +
 +
We let the digestion at 37°C during 3 hours.
 +
 +
===='''2 - Denaturation of EcoRI/SpeI used for digestion of PSB1C3 plasmid and Pfnr '''====
 +
 +
Abdou
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]]
 +
 +
We used 50µL of DNA.
 +
On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!
 +
 +
===='''3 - Ligation of PSB1C3 and Pfnr'''====
 +
 +
Sheng, Zhou
 +
 +
Used quantities :
 +
* Mix A : we mix our digestion mixes :
 +
** Digestion mix of PSB1C3 : 4µL
 +
** Digestion mix of Pfnr : 30µL
 +
** Buffer ligation : 2µL
 +
** H2O : 14µL
 +
 +
* Then, we denature EcoRI/SpeI used for the digestion by ethanol precipitation.
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/Ethanol precipitation|Ethanol precipitation]]
 +
 +
We used 50µL of DNA.
 +
On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!
 +
 +
* Finally we did the ligation mix :
 +
** Buffer ligation : 2µL
 +
** Ligase : 1µL
 +
** DNA : 2µL
 +
** H2O : 15µL
 +
 +
We let the ligation 1h30.
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 13:01, 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

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

2 - Denaturation of EcoRI/SpeI used for digestion of PSB1C3 plasmid and Pfnr

Abdou

Protocol : Ethanol precipitation

We used 50µL of DNA. On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!

3 - Ligation of PSB1C3 and Pfnr

Sheng, Zhou

Used quantities :

  • Mix A : we mix our digestion mixes :
    • Digestion mix of PSB1C3 : 4µL
    • Digestion mix of Pfnr : 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. On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!

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

We let the ligation 1h30.

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