Team:Paris Saclay/Notebook/August/18

From 2014.igem.org

(Difference between revisions)
(→PCR of pCola)
m (→Monday 18th August)
 
(24 intermediate revisions not shown)
Line 2: Line 2:
=Monday 18th August=
=Monday 18th August=
==Lab work==
==Lab work==
 +
===Construction of the fusion protein (color)===
 +
====PCR of the chromoprotein====
 +
''by Mélanie''
 +
 +
{| class="wikitable centre" width="50%"
 +
|+ 
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|H<sub>2</sub>O
 +
|27μl
 +
|-
 +
|5X Phusion buffer
 +
|10µl
 +
|-
 +
|dNTP 10mM
 +
|1µl
 +
|-
 +
|iPS83
 +
|1µl
 +
|-
 +
|iPS84
 +
|1µl
 +
|-
 +
|DNA
 +
|.5µl
 +
|-
 +
|DMSO
 +
|1.5µl
 +
|-
 +
|Phusion
 +
|.5µl
 +
|}
 +
 +
{| class="wikitable centre" width="50%"
 +
|+ PCR cycle
 +
|-
 +
! scope=col | step
 +
! scope=col | temperature (°C)
 +
! scope=col | time (s)
 +
|-
 +
|1
 +
|98
 +
|30
 +
|-
 +
|2
 +
|98
 +
|10
 +
|-
 +
|3
 +
|58
 +
|30
 +
|-
 +
|4
 +
|72
 +
|45
 +
|-
 +
|5
 +
|72
 +
|600
 +
|}
 +
===D - Lemon Scent===
===D - Lemon Scent===
Line 16: Line 79:
! scope=col | volumes
! scope=col | volumes
|-
|-
-
|DNA of GS
+
|DNA of BBa_K517003
|1 μl
|1 μl
|-
|-
Line 22: Line 85:
|10 μl
|10 μl
|-
|-
-
|GoTag enzyme
+
|GoTaq enzyme
|0.5 µl
|0.5 µl
|-
|-
Line 46: Line 109:
Five tubes were prepared. Protocol was modified to match the one used for BBa_K517003 and p cola (PCR conducted on [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/8#PCR 8th August]).  
Five tubes were prepared. Protocol was modified to match the one used for BBa_K517003 and p cola (PCR conducted on [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/8#PCR 8th August]).  
-
Primers used: iPS66 and iPS67.
+
{| class="wikitable centre" width="50%"
 +
|+ PCR of BBa_K762100
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|DNA of BBa_K517003
 +
|1μl
 +
|-
 +
|green GoTaq buffer 5X
 +
|10μl
 +
|-
 +
|GoTaq enzyme
 +
|0.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|31.25µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|4µl
 +
|-
 +
|dNTP
 +
|1µl
 +
|-
 +
|IPS66
 +
|1µl
 +
|-
 +
|IPS67
 +
|1µl
 +
|-
 +
|GoTaq polymerase
 +
|.25µl
 +
|}
====PCR of pCola====
====PCR of pCola====
-
"by Laetitia and Melanie"
+
''by Laetitia and Melanie''
 +
 
 +
We realized a PCR to amplify the geraniol synthase gene.
Five tubes were prepared. Protocol was modified to match the one used for pCola (PCR conducted on [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/8#PCR 8th August]).  
Five tubes were prepared. Protocol was modified to match the one used for pCola (PCR conducted on [https://2014.igem.org/Team:Paris_Saclay/Notebook/August/8#PCR 8th August]).  
-
Primers used : iPS91bis and iPS82
+
{| class="wikitable centre" width="50%"
 +
|+ PCR of BBa_K762100
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|DNA of p cola
 +
|1μl
 +
|-
 +
|green GoTaq buffer 5X
 +
|10μl
 +
|-
 +
|GoTaq enzyme
 +
|0.5µl
 +
|-
 +
|H<sub>2</sub>O
 +
|31.25µl
 +
|-
 +
|MgCl<sub>2</sub>
 +
|4µl
 +
|-
 +
|dNTP
 +
|1µl
 +
|-
 +
|iPS82
 +
|1µl
 +
|-
 +
|iPS91bis
 +
|1µl
 +
|-
 +
|GoTaq polymerase
 +
|.25µl
 +
|}
 +
 
 +
====Electrophoresis of the 3 PCR====
 +
 
 +
 
 +
PS : pinene synthase
 +
 
 +
LS : limonen synthase
 +
 
 +
GS : geraniol synthase
 +
 
 +
PS1 - PS2 - PS3 - PS4 - PS5 - PS6 - LS1 - LS2 - LS3 - LS4 - LS5 - GS1 - GS2 - GS3 - GS4 - GS5 - L
 +
 
 +
[[File:Photo 1808.jpg|300px]]
 +
 
 +
We have PCR product !
 +
 
 +
====Ligation of PCR products inside pGEMTeasy====
 +
''by Laetitia''
 +
 
 +
We used the 3 previously PCR products to perform 3 reaction of ligation. The DNA insert used for each was : PS-6, LS-1 and GS-5
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+ Ligation of BBa_K517003
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|(2X)T4 ligase buffer
 +
|5μL
 +
|-
 +
|pGEMT easy(50ng)
 +
|1 μl
 +
|-
 +
|DNA insert : BBa_K517003 digestion product
 +
|3μL
 +
|-
 +
|Ligase
 +
|1 µl
 +
|}
 +
 
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+ Ligation of BBa_K762100
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|(2X)T4 ligase buffer
 +
|5μL
 +
|-
 +
|pGEMT easy(50ng)
 +
|1 μl
 +
|-
 +
|DNA insert : BBa_K762100 digestion product
 +
|3μL
 +
|-
 +
|Ligase
 +
|1 µl
 +
|}
 +
 
 +
{| class="wikitable centre" width="50%"
 +
|+ Ligation of pCola
 +
|-
 +
! scope=col | components
 +
! scope=col | volumes
 +
|-
 +
|(2X)T4 ligase buffer
 +
|5μL
 +
|-
 +
|pGEMT easy(50ng)
 +
|1 μl
 +
|-
 +
|DNA insert : pCola digestion product
 +
|3μL
 +
|-
 +
|Ligase
 +
|1 µl
 +
|}
 +
 
 +
16°C until afternoon and 4°C during the night
 +
 
 +
==Human Practices==
 +
===Art & Design===
 +
''by Terry''
 +
 
 +
Incubation of a preculture of ''E. Coli'' with plasmid FNR RBS AmylCP (coding for a blue chromoprotein) in our new M63 medium for 24h.
 +
 
 +
==Photo of the Day==
 +
[[File:Paris Saclay 18_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 14:53, 14 October 2014

Contents

Monday 18th August

Lab work

Construction of the fusion protein (color)

PCR of the chromoprotein

by Mélanie

components volumes
H2O 27μl
5X Phusion buffer 10µl
dNTP 10mM 1µl
iPS83 1µl
iPS84 1µl
DNA .5µl
DMSO 1.5µl
Phusion .5µl
PCR cycle
step temperature (°C) time (s)
1 98 30
2 98 10
3 58 30
4 72 45
5 72 600

D - Lemon Scent

PCR of BBa_K517003

By Hoang Vu and Eugène

We realized a PCR to amplify the β-pinene synthase gene.

PCR of BBa_K517003
components volumes
DNA of BBa_K517003 1 μl
green goTaq buffer 5X 10 μl
GoTaq enzyme 0.5 µl
H2O 29.5 µl
MgCl2 4 µl
dNTP 1µl
IPS68bis 2 µl
IPS69bis 2 µl

PCR of BBa_K762100

by Sean

Five tubes were prepared. Protocol was modified to match the one used for BBa_K517003 and p cola (PCR conducted on 8th August).

PCR of BBa_K762100
components volumes
DNA of BBa_K517003 1μl
green GoTaq buffer 5X 10μl
GoTaq enzyme 0.5µl
H2O 31.25µl
MgCl2 4µl
dNTP 1µl
IPS66 1µl
IPS67 1µl
GoTaq polymerase .25µl

PCR of pCola

by Laetitia and Melanie

We realized a PCR to amplify the geraniol synthase gene.

Five tubes were prepared. Protocol was modified to match the one used for pCola (PCR conducted on 8th August).

PCR of BBa_K762100
components volumes
DNA of p cola 1μl
green GoTaq buffer 5X 10μl
GoTaq enzyme 0.5µl
H2O 31.25µl
MgCl2 4µl
dNTP 1µl
iPS82 1µl
iPS91bis 1µl
GoTaq polymerase .25µl

Electrophoresis of the 3 PCR

PS : pinene synthase

LS : limonen synthase

GS : geraniol synthase

PS1 - PS2 - PS3 - PS4 - PS5 - PS6 - LS1 - LS2 - LS3 - LS4 - LS5 - GS1 - GS2 - GS3 - GS4 - GS5 - L

Photo 1808.jpg

We have PCR product !

Ligation of PCR products inside pGEMTeasy

by Laetitia

We used the 3 previously PCR products to perform 3 reaction of ligation. The DNA insert used for each was : PS-6, LS-1 and GS-5

Ligation of BBa_K517003
components volumes
(2X)T4 ligase buffer 5μL
pGEMT easy(50ng) 1 μl
DNA insert : BBa_K517003 digestion product 3μL
Ligase 1 µl


Ligation of BBa_K762100
components volumes
(2X)T4 ligase buffer 5μL
pGEMT easy(50ng) 1 μl
DNA insert : BBa_K762100 digestion product 3μL
Ligase 1 µl
Ligation of pCola
components volumes
(2X)T4 ligase buffer 5μL
pGEMT easy(50ng) 1 μl
DNA insert : pCola digestion product 3μL
Ligase 1 µl

16°C until afternoon and 4°C during the night

Human Practices

Art & Design

by Terry

Incubation of a preculture of E. Coli with plasmid FNR RBS AmylCP (coding for a blue chromoprotein) in our new M63 medium for 24h.

Photo of the Day

Paris Saclay 18 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

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/August/18"