Team:Paris Saclay/Notebook/September/2

From 2014.igem.org

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(→B-Contruction of the fusion protein)
(→D- Lemon scent)
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Limonene synthase
Limonene synthase
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
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
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{| class="wikitable centre" width="50%"
 +
|+
 +
|-
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! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|H<sub>2</sub>O
 +
|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.
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 +
{| class="wikitable centre" width="80%"
 +
|+
 +
|-
 +
! scope=col | Cycle step
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! scope=col | Temperature
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! scope=col | Time
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! scope=col | Cycle
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|-
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| width="25%" |
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Initial denaturation
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| width="25%" |
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98°C
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| width="25%" |
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1 min
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| width="25%" |
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1
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|-
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| Denaturation
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| 98°C
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| 15 s
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| 25 - 30
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|-
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| Annealing
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| 52°C
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| 25 s
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| 25 - 30
 +
|-
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| Extension
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| 72°C
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| 45 s
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| 25-30
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|-
 +
| Final extension
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| 72°C
 +
| 10 min
 +
| 1
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|-
 +
| Final extension
 +
| 8°C
 +
| hold
 +
| 1
 +
|}
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 +
 +
pPS5
 +
To have more plasmid We do some bacteria culture (pPS5)

Revision as of 18:27, 3 September 2014

Labwork

B-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.

D- Lemon scent

By Melanie

Limonene synthase Migration of the PCR done yesterday

photo


well 1= ladder well 2-6 = pPS5 other well = 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:

[photo]

We conclude that we have done a good manipulation.


Limonene synthase 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

98°C

1 min

1

Denaturation 98°C 15 s 25 - 30
Annealing 52°C 25 s 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1


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

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