Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

(Difference between revisions)
(Created page with "{{Team:Paris_Saclay/incl_debut_generique}} ='''Notebook : August 26'''= =='''summary'''== *started the Restriction digestion and ligation to get the BioBrick fnr into plasmid P...")
(→lab work)
Line 17: Line 17:
**''1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3''
**''1.BioBrick promotor fnr(repressor and activator) in plasmid PSB1C3''
<br>
<br>
-
:<u>Digestion for promoter fnr(repressor) in PSB1C3</u>
+
:<u>Digestion for promoter fnr(repressor and activator) in PSB1C3</u>
<br>
<br>
:2 enzymes SPE I and PST I  
:2 enzymes SPE I and PST I  
Line 113: Line 113:
|20µl
|20µl
|}
|}
 +
 +
:For promoter fnr(activator)narG clone 6 :
 +
 +
{| border="1" align="center"
 +
|-o
 +
|DNA
 +
|7µl
 +
|-
 +
| SPE I
 +
|2µl
 +
|-
 +
|PST I
 +
|2µl
 +
|-
 +
|H2O
 +
|7µl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|total
 +
|20µl
 +
|}
 +
 +
:For promoter fnr(activator)nark clone 6 :
 +
 +
{| border="1" align="center"
 +
|-o
 +
|DNA
 +
|4µl
 +
|-
 +
| SPE I
 +
|2µl
 +
|-
 +
|PST I
 +
|2µl
 +
|-
 +
|H2O
 +
|10µl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|total
 +
|20µl
 +
|}
 +
 +
:For promoter fnr(activator)nirB clone 6 :
 +
 +
{| border="1" align="center"
 +
|-o
 +
|DNA
 +
|4µl
 +
|-
 +
| SPE I
 +
|2µl
 +
|-
 +
|PST I
 +
|2µl
 +
|-
 +
|H2O
 +
|10µl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|total
 +
|20µl
 +
|}
 +
 +
:For promoter fnr(activator)nark Digested SPE I :
 +
 +
{| border="1" align="center"
 +
|-oon
 +
|DNA
 +
|4µl
 +
|-
 +
| SPE I
 +
|2µl
 +
|-
 +
|PST I
 +
|2µl
 +
|-
 +
|H2O
 +
|10µl
 +
|-
 +
|buffer
 +
|2µl
 +
|-
 +
|total
 +
|20µl
 +
|}
 +
 +

Revision as of 08:10, 9 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : August 26

summary

  • 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
  • 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 and activator) in plasmid PSB1C3


Digestion for promoter fnr(repressor and activator) in PSB1C3


2 enzymes SPE I and PST I
For promoter fnr(repressor) :
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl


For promoter fnr(activator)nirB clone 6:
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl
For promoter fnr(activator)nark clone 6 :
DNA 4µl
SPE I 2µl
PST I 2µl
H2O 10µl
buffer 2µl
total 20µl
For promoter fnr(activator)nirB clone 7 :
DNA 14µl
SPE I 2µl
PST I 2µl
H2O 0µl
buffer 2µl
total 20µl
For promoter fnr(activator)narG clone 6 :
DNA 7µl
SPE I 2µl
PST I 2µl
H2O 7µl
buffer 2µl
total 20µl
For promoter fnr(activator)nark clone 6 :
DNA 4µl
SPE I 2µl
PST I 2µl
H2O 10µl
buffer 2µl
total 20µl
For promoter fnr(activator)nirB clone 6 :
DNA 4µl
SPE I 2µl
PST I 2µl
H2O 10µl
buffer 2µl
total 20µl
For promoter fnr(activator)nark Digested SPE I :
DNA 4µl
SPE I 2µl
PST I 2µl
H2O 10µl
buffer 2µl
total 20µl





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.


Previous week Back to calendar Next day


Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/26"