Team:Paris Saclay/Notebook/August/6

From 2014.igem.org

(Difference between revisions)
(→PCR Cleanup)
m (→Wednesday 6th August)
 
(41 intermediate revisions not shown)
Line 1: Line 1:
 +
{{Team:Paris_Saclay/notebook_header}}
=Wednesday 6th August=
=Wednesday 6th August=
==Lab work==
==Lab work==
-
===D - Lemon scent===
+
===The chassis coli Odor free===
-
====PCR Cleanup====
+
====Final stock of MG1655====
 +
''by Romain''
 +
 
 +
2 final stocks of the MG1655 made with 1ml of strain and 250µl glycerol 87%, in -80°C.
 +
 
 +
===Salicylate Inducible Suppressing System===
 +
====Plasmid DNA Purification====
 +
''by Eugene and Fabio''
 +
* BBa_K1372000 Cl.1
 +
* BBa_K1372000 Cl.2
 +
From Liquid Culture made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/5#C_-_Salicylate_Inducible_Suppressing_System 5th August]
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_by_using_NucleoSpin®_Tissue Protocol]
 +
 
 +
====Digestion====
 +
''by Eugene and Fabio''
 +
 
 +
In order to place the '''BBa_B0015''' just after the '''BBa_K1372000''', we will start the [http://parts.igem.org/Help:Assembly/3A_Assembly Assembly procedure] again.
 +
* BioBrick '''BBa_K1372000''' (Salicylate promoter + NahR + RNA suppressor) as '''Part A'''
 +
* BioBrick '''BBa_B0015''' (Double terminator) as '''Part B'''
 +
The enzymes used were:
 +
 
 +
* '''EcoRI''' and '''SpeI''' for '''BBa_K1372000'''
 +
* '''EcoRI''' and '''XbaI''' for '''BBa_B0015'''
 +
 
 +
<font color="#F00">TODO: Illustration of the process</font>
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Digest_reaction BioBrick Assembly - Digest Reaction Protocol]
 +
====Segregate Process by Electrophoresis====
 +
 
 +
[[File:Paris_Saclay_060814_cut_gel.jpg|200px|left]]
 +
 
 +
''by Eugene and Fabio''
 +
 
 +
# BBa_B0015 digested by '''EcoRI''' and '''XbaI'''
 +
# BBa_K1372000 digested by '''EcoRI''' and '''SpeI'''
 +
 
 +
 
 +
 
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process BioBrick Assembly - Segregate Process Protocol]
 +
 
 +
===Lemon scent===
 +
====PCR Clean-up====
''by Pierre & Sean''
''by Pierre & Sean''
 +
 +
BioBricks used: '''BBa_K762100''' and '''BBa_K517003''' (PCR perfomed on the [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/4 4th August])
 +
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up Protocol]
 +
 +
====PCR digestion====
 +
''by Pierre & Sean''
 +
 +
 +
'''Protocol'''
 +
 +
For each BioBrick ('''Bba_K517003''' and '''BBa_K762100''')mix the following into a microcentrifuge tube.
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|5.5μl
 +
|-
 +
| PacI
 +
| .5μl
 +
|-
 +
| Fast Digest buffer
 +
| 4μl
 +
|-
 +
| Cleaned-up PCR content
 +
| 30μl
 +
|}
 +
 +
Then bain-marie at 37°C for one hour.
 +
Finally conduct PCR cleanup for both BioBricks, with 15µl of NE buffer.
 +
 +
''We acidentally used PvuI instead of PacI. - SC 6/8/14''
 +
 +
====PCR pCola plasmide (Geranyl Synthase)====
 +
''by melanie''
 +
 +
amplification of geranyl Synthase
 +
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_for_the_genomic_DNA_of_bacteria protocol]
 +
 +
PCR protocol:
 +
98° --> 2min
 +
 +
5 PCR cycle:
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | time
 +
! scope=col | 10sec
 +
! scope=col | 20sec
 +
! scope=col | 45sec
 +
|-
 +
!temperature
 +
|98°
 +
|(depending on the tube - see the gradient)
 +
|72°
 +
|}
 +
 +
25 PCR cycle
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | time
 +
! scope=col | 10sec
 +
! scope=col | 20sec
 +
! scope=col | 45sec
 +
|-
 +
!temperature
 +
|98°
 +
|72°
 +
|72°
 +
|}
 +
 +
and last step: 72° during 10min
 +
 +
but we don't have any results
 +
 +
====Gel electrophoresis====
 +
[[File:Sean&Pierre&Melanie.JPG|400px|right]]
 +
''by Sean,Pierre & Melanie''
 +
 +
Samples used:
 +
# '''BBa_K762100''' (2µl)
 +
# '''BBa_K517003''' (2µl)
 +
# '''p cola'''(12µl)
 +
 +
==Bibliography==
 +
 +
''By Hoang Vu''
 +
 +
Still working on my mission: find more informations about limonene, pinene and citral A and B.I got now a couple of very interessant reviews about limonene and pinene.
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 6_august.jpg|600px|center]]
 +
 +
'''Members present:'''
 +
* Instructors and advisors: Alice.
 +
* Students: Eugene, Fabio, Hoang Vu, Juliette, Melanie, Pierre, Romain, Sean and Terry.
 +
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 14:21, 14 October 2014

Contents

Wednesday 6th August

Lab work

The chassis coli Odor free

Final stock of MG1655

by Romain

2 final stocks of the MG1655 made with 1ml of strain and 250µl glycerol 87%, in -80°C.

Salicylate Inducible Suppressing System

Plasmid DNA Purification

by Eugene and Fabio

  • BBa_K1372000 Cl.1
  • BBa_K1372000 Cl.2

From Liquid Culture made the 5th August

Protocol

Digestion

by Eugene and Fabio

In order to place the BBa_B0015 just after the BBa_K1372000, we will start the [http://parts.igem.org/Help:Assembly/3A_Assembly Assembly procedure] again.

  • BioBrick BBa_K1372000 (Salicylate promoter + NahR + RNA suppressor) as Part A
  • BioBrick BBa_B0015 (Double terminator) as Part B

The enzymes used were:

  • EcoRI and SpeI for BBa_K1372000
  • EcoRI and XbaI for BBa_B0015

TODO: Illustration of the process

BioBrick Assembly - Digest Reaction Protocol

Segregate Process by Electrophoresis

Paris Saclay 060814 cut gel.jpg

by Eugene and Fabio

  1. BBa_B0015 digested by EcoRI and XbaI
  2. BBa_K1372000 digested by EcoRI and SpeI


BioBrick Assembly - Segregate Process Protocol

Lemon scent

PCR Clean-up

by Pierre & Sean

BioBricks used: BBa_K762100 and BBa_K517003 (PCR perfomed on the 4th August)

Protocol

PCR digestion

by Pierre & Sean


Protocol

For each BioBrick (Bba_K517003 and BBa_K762100)mix the following into a microcentrifuge tube.

component volume
H2O 5.5μl
PacI .5μl
Fast Digest buffer 4μl
Cleaned-up PCR content 30μl

Then bain-marie at 37°C for one hour. Finally conduct PCR cleanup for both BioBricks, with 15µl of NE buffer.

We acidentally used PvuI instead of PacI. - SC 6/8/14

PCR pCola plasmide (Geranyl Synthase)

by melanie

amplification of geranyl Synthase

protocol

PCR protocol: 98° --> 2min

5 PCR cycle:

time 10sec 20sec 45sec
temperature 98° (depending on the tube - see the gradient) 72°

25 PCR cycle

time 10sec 20sec 45sec
temperature 98° 72° 72°

and last step: 72° during 10min

but we don't have any results

Gel electrophoresis

Sean&Pierre&Melanie.JPG

by Sean,Pierre & Melanie

Samples used:

  1. BBa_K762100 (2µl)
  2. BBa_K517003 (2µl)
  3. p cola(12µl)

Bibliography

By Hoang Vu

Still working on my mission: find more informations about limonene, pinene and citral A and B.I got now a couple of very interessant reviews about limonene and pinene.

Photo of the Day

Paris Saclay 6 august.jpg

Members present:

  • Instructors and advisors: Alice.
  • Students: Eugene, Fabio, Hoang Vu, Juliette, Melanie, Pierre, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/6"