Team:Paris Saclay/Notebook/August/18

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(B - Construction of the fusion protein (color))
(B - Construction of the fusion protein (color))
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==Lab work==
==Lab work==
===B - Construction of the fusion protein (color)===
===B - Construction of the fusion protein (color)===
-
PCR of the chromoprotein:
+
====PCR of the chromoprotein====
-
Oligo are iPS83 and iPS84
+
 
 +
27 µl H<sub>2</sub>O
 +
 
 +
10µl 5X phusion buffer
 +
 
 +
1µl dNTP 10mM
 +
 
 +
1 µl Primer (iPS83 and iPS84)
 +
 
 +
small amount of DNA
 +
 
 +
1,5µl DMSO
 +
 
 +
0,5µl Phusion
 +
 
 +
 
 +
PCR cycle:
 +
 
 +
98° --> 30 sec
 +
 
 +
98° --> 10 sec
 +
 
 +
58° --> 30 sec
 +
 
 +
72° --> 45 sec
 +
 
 +
72°1 --> 10 min
===D - Lemon Scent===
===D - Lemon Scent===

Revision as of 13:38, 20 August 2014

Contents

Monday 18th August

Lab work

B - Construction of the fusion protein (color)

PCR of the chromoprotein

27 µl H2O

10µl 5X phusion buffer

1µl dNTP 10mM

1 µl Primer (iPS83 and iPS84)

small amount of DNA

1,5µl DMSO

0,5µl Phusion


PCR cycle:

98° --> 30 sec

98° --> 10 sec

58° --> 30 sec

72° --> 45 sec

72°1 --> 10 min

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 GS 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).

Primers used: iPS66 and iPS67.

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).

Primers used : iPS91bis and iPS82

Electrophoresis of the 3 PCR

(image)

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

PCR of BBa_K517003
components volumes
(2X)T4 ligase buffer 5μL
pGEMT easy(50ng) 1 μl
GoTaq enzyme 0.5 µl
DNA insert (GS or LS or PS PCR products) 3μL
Ligase 1 µl

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

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