Team:Paris Saclay/Notebook/September/2

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{{Team:Paris_Saclay/notebook_header}}
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=Tuesday 2nd September=
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==Labwork==
==Labwork==
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===B-Contruction of the fusion protein===
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===Contruction of the fusion protein===
'' By Hv''
'' By Hv''
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We first did a liquid culture in 5 mL of LB.
We first did a liquid culture in 5 mL of LB.
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===D- Lemon scent===  
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===Lemon scent===  
''By Melanie''
''By Melanie''
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Migration of the PCR done [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/1#Limone_synthase yesterday]
Migration of the PCR done [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/1#Limone_synthase yesterday]
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photo
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[[File:PCR pPS5 CAD + PCR LSpGMETe 0209.jpg|500px|center]]
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[[File:PCR clone LS pGMETe (12-25) 0209.jpg|500px|center]]
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picture 1
well 1= ladder
well 1= ladder
well 2-6 = pPS5
well 2-6 = pPS5
other well = LS PCR
other well = LS PCR
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picture 2 = LS PCR
As we see, there are nothing on our gel so we conclude that we failed to clone LS in pGMETeasy
As we see, there are nothing on our gel so we conclude that we failed to clone LS in pGMETeasy
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Plasmid extraction using the plasmid DNA extraction kit and electrophoresis:
Plasmid extraction using the plasmid DNA extraction kit and electrophoresis:
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[[File:Extraction, pPS3 pPS4 0209.jpg|500px]]
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[[File:Extraction, pPS3 pPS4 0209.jpg|500px|center]]
We conclude that we have done a good manipulation.
We conclude that we have done a good manipulation.
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Initial denaturation
Initial denaturation
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| width="25%" |
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98°C
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94°C
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1 min
1 min
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| Denaturation
| Denaturation
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| 94°C
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| 15 s
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| 30 s
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| 25 - 30
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| 25  
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| Annealing
| Annealing
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| 52°C
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| 50°C
| 25 s
| 25 s
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| 25 - 30
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| 25
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| Extension
| Extension
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| 72°C
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| 1 min
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| Final extension
| Final extension
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====pPS5====
====pPS5====
To have more plasmid We do some bacteria culture (pPS5)
To have more plasmid We do some bacteria culture (pPS5)
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==Photo of the Day==
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[[File:Paris Saclay 16_september.jpg|600px|center]]
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{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 12:36, 14 October 2014

Contents

Tuesday 2nd September

Labwork

Contruction of the fusion protein

By Hv

We can see that the blue colonies of E.coli are not blue in a simple petri dish with ampicillin. It's a fail. So, to figure our problem we decided to do a sequencing of our plasmids.

We chose 6 colonies from an Xgal-IPTG petri dish:

- 1 completely blue

- 2 blue with a white ring around the blue

- 3 white colony

PS: we found that there is 2 types of blue colonies: completely blue and blue with a white ring around the blue)

We first did a liquid culture in 5 mL of LB.

Lemon scent

By Melanie

Limonene synthase

Migration of the PCR done yesterday

PCR pPS5 CAD + PCR LSpGMETe 0209.jpg
PCR clone LS pGMETe (12-25) 0209.jpg

picture 1 well 1= ladder well 2-6 = pPS5 other well = LS PCR

picture 2 = LS PCR As we see, there are nothing on our gel so we conclude that we failed to clone LS in pGMETeasy

pPPS5

the photo in the same as previously: We saw a band but we will check another time by PCR to be sure.

pPS3 and pPS4

Plasmid extraction using the plasmid DNA extraction kit and electrophoresis:

Extraction, pPS3 pPS4 0209.jpg

We conclude that we have done a good manipulation.

Limonene synthase PCR

As we saw that we don't have anything for LS, We try to do other PCR from the Biobrick (BBA762100). We use two enzyme: Dream taq and Vent taq

component volume
H2O 42μl
buffer 5μl
dNTPs 1μl
iPS66 1μl
iPS67 1μl
DNA 0.5μl
enzyme 0.5μl

Tubes were placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

94°C

1 min

1

Denaturation 94°C 30 s 25
Annealing 50°C 25 s 25
Extension 72°C 1 min 25
Final extension 72°C 10 min 1
Final extension 8°C hold 1

pPS5

To have more plasmid We do some bacteria culture (pPS5)

Photo of the Day

Paris Saclay 16 september.jpg

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