Team:Paris Saclay/Notebook/August/6

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=Wednesday 6th August=
=Wednesday 6th August=
==Lab work==
==Lab work==
-
===C - Salicylate Inducible Suppressing System===
+
===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====
====Plasmid DNA Purification====
''by Eugene and Fabio''
''by Eugene and Fabio''
Line 10: Line 16:
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_by_using_NucleoSpin®_Tissue Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Extraction_of_the_Genomic_DNA_from_Bacteria_by_using_NucleoSpin®_Tissue Protocol]
 +
====Digestion====
====Digestion====
''by Eugene and Fabio''
''by Eugene and Fabio''
Line 25: Line 32:
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Digest_reaction BioBrick Assembly - Digest Reaction Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Digest_reaction BioBrick Assembly - Digest Reaction Protocol]
====Segregate Process by Electrophoresis====
====Segregate Process by Electrophoresis====
 +
 +
[[File:Paris_Saclay_060814_cut_gel.jpg|200px|left]]
 +
''by Eugene and Fabio''
''by Eugene and Fabio''
-
# BBa_K1372000 digested by '''EcoRI''' and '''SpeI'''
 
# BBa_B0015 digested by '''EcoRI''' and '''XbaI'''
# BBa_B0015 digested by '''EcoRI''' and '''XbaI'''
 +
# BBa_K1372000 digested by '''EcoRI''' and '''SpeI'''
-
'''Results:'''
 
-
#
 
-
#
 
-
''In progress''
 
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process BioBrick Assembly - Segregate Process Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process BioBrick Assembly - Segregate Process Protocol]
-
===D - Lemon scent===
+
===Lemon scent===
-
====PCR Cleanup====
+
====PCR Clean-up====
''by Pierre & Sean''
''by Pierre & Sean''
Line 45: Line 51:
[https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up Protocol]
[https://2014.igem.org/Team:Paris_Saclay/Protocols/PCR_clean-up Protocol]
-
 
-
====Gel electrophoresis====
 
-
''by Melanie''
 
-
 
-
Plasmid used: p cola
 
-
 
-
Results inconclusive. Wrong enzyme was used to prepare PCR.
 
====PCR digestion====
====PCR digestion====
''by Pierre & Sean''
''by Pierre & Sean''
 +
'''Protocol'''
'''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==
==Bibliography==
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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.
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:'''
'''Members present:'''

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"