Team:Paris Saclay/Notebook/August/25

From 2014.igem.org

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==Lab work==
==Lab work==
-
===B - Construction of the fusion protein===
+
===Construction of the fusion protein===
====plasmid extraction : pGEMTeasy + chromoprotein ====
====plasmid extraction : pGEMTeasy + chromoprotein ====
Line 48: Line 48:
T : control negatif without enzyme
T : control negatif without enzyme
-
D : sample digested by the two enzymes
+
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|blue
 +
|2 μl
 +
|-
 +
|Plasmid
 +
|1 μl
 +
|-
 +
|H20
 +
|9μl
 +
|}
-
T1 - D1 - T2 - D2 - T3 - D3 - E - T4 - P4 - T5 - o - P6
 
-
[[File:250814 laetitia digestion verif chromoP pGEMTeasy.jpg|500px|]]
+
[[File:250814 laetitia digestion verif chromoP pGEMTeasy.jpg|400px|right]]
-
Results : the samples 3, 4 and 6 seems to contain the chromoprotein gene (two bands)
+
D : sample digested by XbaI and PstI
 +
 
 +
T1 - D1 - T2 - D2 - T3 - D3 - E - T4 - D4 - T5 - o - D6
 +
 
 +
 
 +
Results : lost of the sample D5 in a hole in the agarose gel
 +
 
 +
The samples 3, 4 and 6 seems to contain the chromoprotein gene (two bands)
 +
 
 +
--> We will try another plasmid extraction because the electrophoresis profil doesn't show bands for every sample.
==== Bacterial culture of DH5a containing pGEMTeasy + chromprotein ====
==== Bacterial culture of DH5a containing pGEMTeasy + chromprotein ====
Line 60: Line 82:
We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction
We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction
-
 
===D - Lemon Scent===
===D - Lemon Scent===
====Gel electrophoresis of CAD====
====Gel electrophoresis of CAD====
 +
 +
[[File:Paris_Saclay_Sean_140825.jpg|left]]
 +
''by Sean''
''by Sean''
 +
 +
Veirfication of the purification from [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/22#PCR_of_CAD Friday]
''Legend''
''Legend''
Line 70: Line 96:
# PCR result of CAD
# PCR result of CAD
-
[[File:Paris_Saclay_Sean_140825.jpg]]
+
 
 +
 
 +
==== CAD ligation  in pPS1 ====
 +
''by Mélanie''
 +
pPSI was previously digest by PacI and dephosphorylated
 +
We want to insert the CAD PCR fragment in pPSI to make pPS5.
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|PCR CAD
 +
|10 μl
 +
|-
 +
|pPS1
 +
|3μl
 +
|-
 +
|10x buffer ligase
 +
|2μl
 +
|-
 +
|ligase
 +
|1μl
 +
|-
 +
|H20
 +
|4μl
 +
|}
 +
 
 +
4H at room temperature
 +
 
 +
==== Transformation of DH5a with the ligation ====
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/Transformation_of_supercompetent_Ecoli_cells_with_CaCl2 protocol]
 +
 
 +
 
 +
====Culture of bacteria  with pPS2 - pPS3 - pPS4====
 +
I take some colonies made [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/22#Transformation_in_competent_E.coli_DH5.CE.B1 Friday 22th August]
 +
 
 +
=== Limonene synthase digestion===
 +
 
 +
Because we don't success to have pPS2 - we try to digest again Limonene synthase in pGMET esay
 +
 
 +
{| class="wikitable centre" width="25%"
 +
|+ 
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|LS pGMET esay
 +
|50μl
 +
|-
 +
|FastDigest green buffer 10X
 +
|5μl
 +
|-
 +
|PacI
 +
|0.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|4.5µl
 +
|}
 +
 
 +
when I done the electrophoresis to check the digestion, I have a surprise : no digestion.
 +
 
 +
So we check the pGMETesay plasmid with normaly the inserts (for LS GS and PS)and we saw that there is no insert in the plasmid.
 +
 
 +
 
 +
==Photo of the Day==
 +
[[File:Paris Saclay 25_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 15:14, 14 October 2014

Contents

Monday 25th August

Lab work

Construction of the fusion protein

plasmid extraction : pGEMTeasy + chromoprotein

by Laetitia

We used the bacteria containing pGEMTeasy + chromoprotein launched by Melanie sunday the 24th for the plasmid extraction, 6 independent cultures which come from 6 independent colonies.

We made a stock of bacteria for each sample (x6).

To extract the plasmid, we used the plasmid DNA purification kit (Macherey-Nagel).

Digestion of pGEMTeasy+chromoproteinby XbaI and PstI

by Laetitia

The goal is to check the presence of the insert inside the plasmid. We digest the 6 samples of purified plasmid.

component volume
Plasmid 5 μl
Fast Digest buffer 10X 1μl
XbaI 0.5μl
PstI 0.5μl
H20 3μl

1h at 37°C

Electrophoresis of the digestion product of pGEMTeasy+chromoprotein

by Laetitia

T : control negatif without enzyme

component volume
blue 2 μl
Plasmid 1 μl
H20 9μl


250814 laetitia digestion verif chromoP pGEMTeasy.jpg

D : sample digested by XbaI and PstI

T1 - D1 - T2 - D2 - T3 - D3 - E - T4 - D4 - T5 - o - D6


Results : lost of the sample D5 in a hole in the agarose gel

The samples 3, 4 and 6 seems to contain the chromoprotein gene (two bands)

--> We will try another plasmid extraction because the electrophoresis profil doesn't show bands for every sample.

Bacterial culture of DH5a containing pGEMTeasy + chromprotein

by Laetitia

We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction

D - Lemon Scent

Gel electrophoresis of CAD

Paris Saclay Sean 140825.jpg

by Sean

Veirfication of the purification from Friday

Legend

  1. ladder 10µl
  2. PCR result of CAD


CAD ligation in pPS1

by Mélanie pPSI was previously digest by PacI and dephosphorylated We want to insert the CAD PCR fragment in pPSI to make pPS5.

component volume
PCR CAD 10 μl
pPS1 3μl
10x buffer ligase 2μl
ligase 1μl
H20 4μl

4H at room temperature

Transformation of DH5a with the ligation

protocol


Culture of bacteria with pPS2 - pPS3 - pPS4

I take some colonies made Friday 22th August

Limonene synthase digestion

Because we don't success to have pPS2 - we try to digest again Limonene synthase in pGMET esay

components volumes
LS pGMET esay 50μl
FastDigest green buffer 10X 5μl
PacI 0.5µl
H2O 4.5µl

when I done the electrophoresis to check the digestion, I have a surprise : no digestion.

So we check the pGMETesay plasmid with normaly the inserts (for LS GS and PS)and we saw that there is no insert in the plasmid.


Photo of the Day

Paris Saclay 25 august.jpg

Members present:

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

Back to the calendar

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