Team:Paris Saclay/Notebook/August/29

From 2013.igem.org

(Difference between revisions)
(→3 -Gel electrophoresis of Degestion product of .)
(→lab work)
Line 216: Line 216:
*Gel : 1.0%
*Gel : 1.0%
|}
|}
 +
 +
====1 - Colony PCR of Gibson assembly.====
 +
 +
 +
 +
Colonies repiquée dans 10µL d'eau.
 +
We do 8 PCR for each ligation.
 +
 +
PCR MIX for 55 tubes:
 +
 +
* Buffer Dream Taq : 137.5µL
 +
* dNTP : 27.5µL
 +
* Oligo 44 : 27.5µL
 +
* Oligo 43: 27.5µL
 +
* Dream Taq : 11µL
 +
* H2O : 1.144mL
 +
 +
 +
 +
Add 2µL bacteria into 23µL PCR MIX in PCR tube
 +
PCR Program
 +
 +
[[File:PsPCRLacZ2108.jpg|400px]]
 +
 +
 +
{| border="1" align="center"
{| border="1" align="center"
||<big>Previous week</big>
||<big>Previous week</big>

Revision as of 14:59, 19 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 29

summary

  • We got clonies 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 clonies 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


Digestion for PSB3K3



DNA 9µl
Ecoil I 2µl
PST I 2µl
H2O 5µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_10(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_11(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_LacZ+Term_15(08/09/13)

B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3


DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl


Digestion for RBS_AmilCP+Term_9(02/09/13)



DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Digestion for RBS_AmilCP+Term_12(02/09/13)



DNA 14µl
xpe I 2µl
PST I 2µl
buffer 2µl
total 20µl
Degestion product quantification
We used the nano drop to measure the DNA in 260nm and we found its concentration
name fnr(repressor)dig EcoilI and speI fnr nark dig EcoilI and speI
conc. 25.0ng/µl 13.6ng/µl




2 -Gel electrophoresis of Degestion product of promoter fnr(activator).

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of promoter fnr(activator)nirB clone 7 dig SPE I and PstI
  • Well 3 : 3µL of promoter fnr(activator)narG clone 6 dig SPE I and PstI
  • Well 4 : 3µL of promoter fnr(activator)nark Digested SPE I and PstI
  • Well 5: 3µL of promoter fnr(activator)narB Digested SPE I and PstI
  • Well 6: 3µL of promoter fnr(activator)narG Digested SPE I and PstI


  • Gel : 1.0%


3 -Gel electrophoresis of Degestion product of RBS_AmilCP+Term and RBS_LacZ+Term.

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of RBS_AmilCP+Term_9 digested by xpe I and PstI
  • Well 3 : 3µL of RBS_AmilCP+Term_12 digested by xpe I and PstI
  • Well 4 : 3µL of RBS_LacZ+Term_10 digested by xpe I and PstI
  • Well 5: 3µL of RBS_LacZ+Term_11 digested by xpe I and PstI
  • Well 6: 3µL of RBS_LacZ+Term_15 digested by xpe I and PstI
  • Gel : 1.0%

1 - Colony PCR of Gibson assembly.

Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.

PCR MIX for 55 tubes:

  • Buffer Dream Taq : 137.5µL
  • dNTP : 27.5µL
  • Oligo 44 : 27.5µL
  • Oligo 43: 27.5µL
  • Dream Taq : 11µL
  • H2O : 1.144mL


Add 2µL bacteria into 23µL PCR MIX in PCR tube PCR Program

PsPCRLacZ2108.jpg


Previous week Back to calendar Next day


Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/29"