Team:Paris Saclay/Notebook/August/27

From 2013.igem.org

(Difference between revisions)
(→summary)
(→summary)
Line 4: Line 4:
=='''summary'''==  
=='''summary'''==  
-
*We got clonies on ligation promoter fnr(activator)nirB plus  RBS_LacZ+Term_PSB1C3   
+
*We got colonies on ligation promoter fnr(activator)nirB plus  RBS_LacZ+Term_PSB1C3   
promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3   
promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3   
promoter fnr(repressor) plus  RBS_LacZ+Term_PSB1C3  
promoter fnr(repressor) plus  RBS_LacZ+Term_PSB1C3  
promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3t
promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3t
so we do Colony PCR for it
so we do Colony PCR for it
-
* We didn't get clonies on Gibson so we do a gel electrophoresis of  parts of Gibson assembly to verify the size
+
* We didn't get colonies on Gibson so we do a gel electrophoresis of  parts of Gibson assembly to verify the size
* Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
* Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
<br>
<br>

Revision as of 12:02, 21 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 27

summary

  • We got colonies on ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3

promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3t so we do Colony PCR for it

  • We didn't get colonies on Gibson so we do a gel electrophoresis of parts of Gibson assembly to verify the size
  • Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and electrophoresis of it .


lab work


  • A.aero/anaerobic regulation system
    • 1.Colony PCR on e.coli with FNR Promoter plus RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+Term_PSB1C3 for 8 colonies of each

Xiaojing

  • A1-8: promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3
  • B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
  • C1-8: promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3
  • D1-8: promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
  • Picking of 32 colonies
  • Preparation Master mix
    • H2O : 490µL
    • dNTP : 17.5µL
    • VR primer : 17.5µL
    • VF2 primer : 17.5µL
    • DreamTaq buffer 10x : 87.5µL
    • DreamTaq enzyme : 7µL

Protocol : Colony PCR PCR Program :

PsPcr808.jpg




2 - Gel electrophoresis of the colony PCR products
[[]]
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp

A - Aerobic/Anaerobic regulation system / B - PCB sensing system

1 -Gel electrophoresis of parts of Gibson assembly.

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 1µL of PSB1C3 Clean
  • Well 3 : 3µL of RBS_BphR2 part1+1µl of 6X loading dy
  • Well 4 : 3µL of BphR2 part2+1µl of 6X loading dy
  • Well 5: 3µL of RBS_FNR part1 +1µl of 6X loading dy
  • Well 6: 3µL of FNR part1 +1µl of 6X loading dy
  • Well 7 : 3µL of FNR part2 +1µl of 6X loading dy
  • Gel : 1.0% 
Now we do a Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson.
PSB1C3 clean for Gibson 17µl
enzyme Dnp1 1µl
buffer 2µl
total 20µl
Incubate at 37°C for 1h30mins.
2 - Gel purification of the PSB1C3 Clean for Gibson

2.1 - Migration of 20µl PSB1C3 Clean for Gibson digested by Dnp1 '

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 1µL of PSB1C3 Clean dig Dnp1(08/06/2013)
  • Well 3 : 20µL of PSB1C3 Clean dig Dnp1(08/27/2013)


  • Gel : 1.0%


Previous week Back to calendar Next day


Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/27"