Team:Paris Saclay/Notebook/August/27

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=Wednesday 27th August=
=Wednesday 27th August=
==Lab Work==
==Lab Work==
-
===D - lemon scent ===
+
===Construction of the fusion protein (color)===
 +
 
 +
==== Electrophoresis of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI ====
 +
[[File:270814 Laetitia gel decoup chromo.jpg|300px|left]]
 +
''by Laetitia''
 +
 
 +
Digestion made the 26th by Laetitia. After the migration on agarose gel (100V), we cut the band corresponding to the chromoprotein gene on UV table.
 +
 
 +
 
 +
 
 +
====Purification of the chromoprotein gene from the agarose gel ====
 +
''by Sean''
 +
 
 +
We used the kit PCR-Clean-UP to extract the DNA from the agarose gel. Elution volume : 15µl
 +
 
 +
====Gel electrophoresis of the purification product ====
 +
''by Sean''
 +
 
 +
The goal is to check if the purification of the chromoprotein gene has worked.
 +
 
 +
PCR clean-up was performed ealier [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/27#PCR_clean-up_of_colonies_containing_pGEMTeasy.2Bchromoprotein today]. 1µl of each result was used for the electrophoresis. Nothing appeared under UV.
 +
 
 +
====Ligation of chromoprotein====
 +
''by Sean''
 +
 
 +
{| class="wikitable centre" width="20%"
 +
|+
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|H<sub>2</sub>O
 +
|6μl
 +
|-
 +
|T4 DNA ligase buffer
 +
|2µl
 +
|-
 +
|purified chromoprotein
 +
|10µl
 +
|-
 +
|ligase
 +
|1µl
 +
|-
 +
|pSB1C3
 +
|1µl
 +
|}
 +
 
 +
Leave overnight at 16°C.
 +
 
 +
====Collection of pellets containing pGEMTeasy+chromoprotein====
 +
''by Sean''
 +
 
 +
Three liquid cultures (pGEM+chromoprotein) were prepared. 2ml from each were collected in 2ml microcentrifuge tubes, then centrifuged. The pellets were put in the freezer.
 +
 
 +
==== Transformation of odor free E. coli with plasmids coding Fluo Protein ====
 +
''by Terry''
 +
 
 +
Our fusion protein was not expressed in pGEMTeasy (no color in the culture), so, if our system do not work at all, I'm preparing FP (Fluorescent Protein) for our lemon.
 +
 
 +
6 plamids coding FP have been transformed in Odor Free :
 +
*pCFP+
 +
*pYFP+
 +
*pRFP+
 +
*pEGFP
 +
*pGFP
 +
*pEYFP
 +
 
 +
===lemon scent ===
====Plasmide extraction====
====Plasmide extraction====
'' By Mélanie''
'' By Mélanie''
Line 8: Line 75:
Extraction of pPS5 with the phenol protocol (as previously described)
Extraction of pPS5 with the phenol protocol (as previously described)
 +
====PCR of Limonene synthase (BBa762100)====
 +
''by Mélanie''
 +
 +
5 tubes to do a gradient
 +
 +
{| class="wikitable centre" width="50%"
 +
|+ 
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|H<sub>2</sub>O
 +
|29.75μl
 +
|-
 +
|Gotaq buffer
 +
|10µl
 +
|-
 +
|dNTP 10mM
 +
|1µl
 +
|-
 +
|iPS67
 +
|1µM
 +
|-
 +
|iPS66
 +
|1µM
 +
|-
 +
|DNA
 +
|1µl
 +
|-
 +
|MgCl2
 +
|4µl
 +
|-
 +
|Gotaq
 +
|1.25µl
 +
|}
 +
 +
{| class="wikitable centre" width="50%"
 +
|+ PCR cycle
 +
|-
 +
! scope=col | step
 +
! scope=col | temperature (°C)
 +
! scope=col | time
 +
|-
 +
|1
 +
|95
 +
|2min
 +
|-
 +
|2
 +
|95
 +
|30 sec
 +
|-
 +
|3
 +
|49-55 (gradient)
 +
|30sec
 +
|-
 +
|4
 +
|72
 +
|2min
 +
|-
 +
|5
 +
|72
 +
|5min
 +
|}
 +
 +
==== Electrophoresis of pPS3 and pPS4====
 +
 +
[[File:Paris_Saclay_electrophoresis_pPS3_pPS4.jpeg|400px|right]]
 +
 +
''by Terry''
 +
 +
Result from [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/26 yesterday] 's extraction.
 +
 +
Gel shaft :
 +
 +
*0 Ladder
 +
*1 pPS4 (geraniol synthase)
 +
*2 pPS3 (pinene synthase)
 +
*3 pPS3
 +
*4 pPS4
 +
*5 pPS4
 +
*6 pPS4
 +
*7 pPS4
 +
*8 Ladder
 +
 +
 +
The pPS4 (shaft 4) seems to correspond to our reference but the gel is not top quality. More analysis need to be made.
 +
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 27_august.jpg|600px|center]]
 +
 +
'''Members present:'''
 +
* Instructors and advisors: Alice, Solenne and Sylvie.
 +
* Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:20, 14 October 2014

Contents

Wednesday 27th August

Lab Work

Construction of the fusion protein (color)

Electrophoresis of pGEMTeasy+chromoprotein 3,4 and 6 by EcoRI and PstI

270814 Laetitia gel decoup chromo.jpg

by Laetitia

Digestion made the 26th by Laetitia. After the migration on agarose gel (100V), we cut the band corresponding to the chromoprotein gene on UV table.


Purification of the chromoprotein gene from the agarose gel

by Sean

We used the kit PCR-Clean-UP to extract the DNA from the agarose gel. Elution volume : 15µl

Gel electrophoresis of the purification product

by Sean

The goal is to check if the purification of the chromoprotein gene has worked.

PCR clean-up was performed ealier today. 1µl of each result was used for the electrophoresis. Nothing appeared under UV.

Ligation of chromoprotein

by Sean

components volumes
H2O 6μl
T4 DNA ligase buffer 2µl
purified chromoprotein 10µl
ligase 1µl
pSB1C3 1µl

Leave overnight at 16°C.

Collection of pellets containing pGEMTeasy+chromoprotein

by Sean

Three liquid cultures (pGEM+chromoprotein) were prepared. 2ml from each were collected in 2ml microcentrifuge tubes, then centrifuged. The pellets were put in the freezer.

Transformation of odor free E. coli with plasmids coding Fluo Protein

by Terry

Our fusion protein was not expressed in pGEMTeasy (no color in the culture), so, if our system do not work at all, I'm preparing FP (Fluorescent Protein) for our lemon.

6 plamids coding FP have been transformed in Odor Free :

  • pCFP+
  • pYFP+
  • pRFP+
  • pEGFP
  • pGFP
  • pEYFP

lemon scent

Plasmide extraction

By Mélanie

Extraction of pPS5 with the phenol protocol (as previously described)

PCR of Limonene synthase (BBa762100)

by Mélanie

5 tubes to do a gradient

components volumes
H2O 29.75μl
Gotaq buffer 10µl
dNTP 10mM 1µl
iPS67 1µM
iPS66 1µM
DNA 1µl
MgCl2 4µl
Gotaq 1.25µl
PCR cycle
step temperature (°C) time
1 95 2min
2 95 30 sec
3 49-55 (gradient) 30sec
4 72 2min
5 72 5min

Electrophoresis of pPS3 and pPS4

Paris Saclay electrophoresis pPS3 pPS4.jpeg

by Terry

Result from yesterday 's extraction.

Gel shaft :

  • 0 Ladder
  • 1 pPS4 (geraniol synthase)
  • 2 pPS3 (pinene synthase)
  • 3 pPS3
  • 4 pPS4
  • 5 pPS4
  • 6 pPS4
  • 7 pPS4
  • 8 Ladder


The pPS4 (shaft 4) seems to correspond to our reference but the gel is not top quality. More analysis need to be made.


Photo of the Day

600px

Members present:

  • Instructors and advisors: Alice, Solenne and Sylvie.
  • Students: Eugène, Hoang Vu, Laëtitia, Mélanie, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/27"