Team:Paris Saclay/Notebook/September/3

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==B- Construction of the fusion protein==
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{{Team:Paris_Saclay/notebook_header}}
 +
=Wednesday 3rd September=
 +
 
 +
==LabWork==
 +
===Construction of the fusion protein===
We made a classic exttraction of plasmids from the liquid cultures.
We made a classic exttraction of plasmids from the liquid cultures.
Check by electrophoresis if it worked and send it for sequencing.
Check by electrophoresis if it worked and send it for sequencing.
 +
 +
 +
===Lemon scent===
 +
''by Mélanie''
 +
 +
====PCR LS====
 +
 +
Electrophoresis of the PCR made [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/2#Limonene_synthase_PCR Yesterday]
 +
 +
[[File:0309 PCR LS + extraction pPS5.jpg|400px|center]]
 +
 +
well 2-3 = PCR with Dream taq or Vent taq
 +
 +
(well 4-5 = checking of the pps5 Extraction)
 +
 +
 +
The PCR have success so I use the PCR clean up kit to purify it
 +
 +
Electrophoresis after purification
 +
 +
[[File:0309 purif LS.jpg|400px|center]]
 +
 +
 +
====Digestion====
 +
In order to clone LS in pPSI, i Digest it by PacI
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|3μl
 +
|-
 +
|buffer
 +
|1μl
 +
|-
 +
|PacI
 +
|1μl
 +
|-
 +
|PCR purify
 +
|5μl
 +
|}
 +
 +
====Ligation====
 +
We already have some pPSI digested and dephosphorelated
 +
 +
So I do a ligation:
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|5μl
 +
|-
 +
|buffer
 +
|2μl
 +
|-
 +
|ligase
 +
|1μl
 +
|-
 +
|pPSI
 +
|2μl
 +
|-
 +
|LS PCR
 +
|10μl
 +
|}
 +
 +
2 hours at room temperature and over night at 4°
 +
 +
====pPS5====
 +
Plasmid exctraction using the kit (picture is [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/3#PCR_LS here])
 +
 +
digestion by SalI
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|11μl
 +
|-
 +
|buffer
 +
|5μl
 +
|-
 +
|SalI
 +
|2μl
 +
|-
 +
|pPS5
 +
|30μl
 +
|}
 +
Electrophoresis
 +
 +
[[File:0309 digestion SalI pPS5.jpg|500px|center]]
 +
 +
We saw that we have something strang with our plasmid but we wil check it tomorrow with a good ladder (the ladder here was normally on the well 1)
 +
 +
====pPS3 and pPS4====
 +
We digest the plasmid by HindIII to direct our insert
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|6μl
 +
|-
 +
|buffer
 +
|1μl
 +
|-
 +
|HindIII
 +
|1μl
 +
|-
 +
|pPS3/4
 +
|2μl
 +
|}
 +
 +
[[File:0309 digestion HindIII pPS3 4.jpg|500px|center]]
 +
 +
well 1 = ladder
 +
well 2-3-4 = pPS3
 +
Well 5-6-7 = pPS4
 +
 +
results are very strange
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 3_september.jpg|250px|center]]
 +
 +
[http://iramis.cea.fr/llb/Phocea/Pisp/visu.php?id=35&uid=%20alexei.grinbaum Alexei Grinbaum] , one of those few we have interviewed during this month of september.
 +
 +
To learn more about the ethic aspect of our project, just click [https://2014.igem.org/Team:Paris_Saclay/Ethics here]
 +
 +
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 12:50, 14 October 2014

Contents

Wednesday 3rd September

LabWork

Construction of the fusion protein

We made a classic exttraction of plasmids from the liquid cultures.

Check by electrophoresis if it worked and send it for sequencing.


Lemon scent

by Mélanie

PCR LS

Electrophoresis of the PCR made Yesterday

0309 PCR LS + extraction pPS5.jpg

well 2-3 = PCR with Dream taq or Vent taq

(well 4-5 = checking of the pps5 Extraction)


The PCR have success so I use the PCR clean up kit to purify it

Electrophoresis after purification

0309 purif LS.jpg


Digestion

In order to clone LS in pPSI, i Digest it by PacI

component volume
H2O 3μl
buffer 1μl
PacI 1μl
PCR purify 5μl

Ligation

We already have some pPSI digested and dephosphorelated

So I do a ligation:

component volume
H2O 5μl
buffer 2μl
ligase 1μl
pPSI 2μl
LS PCR 10μl

2 hours at room temperature and over night at 4°

pPS5

Plasmid exctraction using the kit (picture is here)

digestion by SalI

component volume
H2O 11μl
buffer 5μl
SalI 2μl
pPS5 30μl

Electrophoresis

0309 digestion SalI pPS5.jpg

We saw that we have something strang with our plasmid but we wil check it tomorrow with a good ladder (the ladder here was normally on the well 1)

pPS3 and pPS4

We digest the plasmid by HindIII to direct our insert

component volume
H2O 6μl
buffer 1μl
HindIII 1μl
pPS3/4 2μl
0309 digestion HindIII pPS3 4.jpg

well 1 = ladder well 2-3-4 = pPS3 Well 5-6-7 = pPS4

results are very strange

Photo of the Day

Paris Saclay 3 september.jpg

[http://iramis.cea.fr/llb/Phocea/Pisp/visu.php?id=35&uid=%20alexei.grinbaum Alexei Grinbaum] , one of those few we have interviewed during this month of september.

To learn more about the ethic aspect of our project, just click here


Back to the calendar

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