Team:Paris Saclay/Notebook/July/1

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='''Notebook : July 1'''=
='''Notebook : July 1'''=
-
=='''Lab work'''==
+
=='''summary'''==
-
constructing
+
 
 +
*Commenced the Restriction digest and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoR I and PST I.
 +
 
 +
*Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which is created by iGEM UCL team in 2009
 +
 
 +
*Designed oligopeptides for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
 +
<br>
 +
 
 +
=='''detail'''==
 +
 
 +
<br>
 +
*'''A.aero/anaerobic regulation system'''<br>
 +
**''1.BioBrick promotor fnr(repressor) in plasmid PSB1C3''
 +
<br>
 +
:<u>Digestion for fnr and PSB1C3</u>
 +
<br>
 +
:2 enzymes EcoR I and PST I can be used in one common buffer: orange buffer (10X).
 +
 
 +
:For PCR products:
 +
 
 +
{| border="1" align="center"
 +
|-
 +
|PCR products
 +
|20µl
 +
|-
 +
|EcoR I
 +
|0.75µl
 +
|-
 +
|PST I
 +
|0.75µl
 +
|-
 +
|H2O
 +
|5.5µl
 +
|-
 +
|buffer
 +
|3µl
 +
|-
 +
|total
 +
|30µl
 +
|}
 +
 
 +
 
 +
:For plasmid PSB1C3:
 +
 
 +
{|align="center" border="1"
 +
|-
 +
|Plasmid
 +
|4µl
 +
|-
 +
|EcoR I
 +
|0.5µl
 +
|-
 +
|PST I
 +
|0.5µl
 +
|-
 +
|H2O
 +
|2.2µl
 +
|-
 +
|buffer
 +
|0.8µl
 +
|-
 +
|total
 +
|8µl
 +
|}
 +
<br>
 +
 
 +
:<u>Ligation</u>
 +
 
 +
:After 3h of digestion, we mixed the digestion products:<br>
 +
 
 +
{|align="center" border="1"
 +
|-
 +
|PCR product
 +
|30µl
 +
|-
 +
|PSB1C3
 +
|4µl
 +
|-
 +
|Ligation buffer
 +
|2µl
 +
|-
 +
|H2O
 +
|14µl
 +
|}<br>
 +
 
 +
:Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
 +
 
 +
{| align="center" border="1"
 +
|-
 +
|mixture
 +
|control
 +
|-
 +
|2µl ligation buffer
 +
|2µl ligation buffer
 +
|-
 +
|1µl ligase T4
 +
|1µl ligase T4+2µl PSB1C3
 +
|-
 +
|17µl H2O
 +
|15µl H2O
 +
|}
 +
<br>
 +
:The incubation was during 1H30.
 +
 
 +
 
 +
*'''B.PCBs sensor system'''<br>
 +
**''1.BioBrick BphR2(regulator) in plasmid PSB1C3''<br>
 +
 
 +
 
 +
:Using software gene manager to find the oligopeptide for amplification of BphR2.
 +
 
 +
 
 +
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 11:50, 21 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : July 1

summary

  • Commenced the Restriction digest and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoR I and PST I.
  • Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which is created by iGEM UCL team in 2009
  • Designed oligopeptides for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group


detail


  • 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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