Team:Paris Saclay/Notebook/August/18

From 2014.igem.org

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(→Ligation of PCR products inside pGEMTeasy)
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====PCR of the chromoprotein====
====PCR of the chromoprotein====
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27 µl H<sub>2</sub>O
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{| class="wikitable centre" width="50%"
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|+ 
 +
|-
 +
! scope=col | components
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! scope=col | volumes
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|-
 +
|H<sub>2</sub>O
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|27μl
 +
|-
 +
|5X Phusion buffer
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|10µl
 +
|-
 +
|dNTP 10mM
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|1µl
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|-
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|iPS83
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|1µl
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|-
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|iPS84
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|1µl
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|-
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|DNA
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|.5µl
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|-
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|DMSO
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|1.5µl
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|-
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|Phusion
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|.5µl
 +
|}
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10µl 5X phusion buffer
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{| class="wikitable centre" width="50%"
-
 
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|+ PCR cycle
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1µl dNTP 10mM
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|-
-
 
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! scope=col | step
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1 µl Primer (iPS83 and iPS84)
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! scope=col | temperature (°C)
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! scope=col | time (s)
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small amount of DNA
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|-
-
 
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|1
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1,5µl DMSO
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|98
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|30
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0,5µl Phusion
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|-
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|2
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|98
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PCR cycle:
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|10
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|-
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98° --> 30 sec
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|3
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|58
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98° --> 10 sec
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|30
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|-
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58° --> 30 sec
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|4
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|72
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72° --> 45 sec
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|45
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|-
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72°1 --> 10 min
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|5
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|72
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|600
 +
|}
===D - Lemon Scent===
===D - Lemon Scent===

Revision as of 09:51, 22 August 2014

Contents

Monday 18th August

Lab work

B - Construction of the fusion protein (color)

PCR of the chromoprotein

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 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
DNA insert (GS or LS or PS PCR products) 3μL
Ligase 1 µl

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

Back to the calendar

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