Team:Paris Saclay/Notebook/September/8

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{{Team:Paris_Saclay/notebook_header}}
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=Monday 8th September=
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==Lab Work==
==Lab Work==
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''by Mélanie''
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===D-Lemon Scent===
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===Lemon Scent===
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====Checking PCR====
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Electrophoresis of the PCR made [http://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#PCR_LS_PS_GS_Cad Friday]
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[photo]
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====PCR with bacteria====
====PCR with bacteria====
From the petri dish ([http://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#Transformation made here], I select some clone to do a PCR :
From the petri dish ([http://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#Transformation made here], I select some clone to do a PCR :
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clones 4-5 for PS
clones 4-5 for PS
clones 6 for LS
clones 6 for LS
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{| class="wikitable centre" width="50%"
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|+
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|-
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! scope=col | component
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! scope=col | volume
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|-
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|H<sub>2</sub>O
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|41.5μl
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|-
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|buffer
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|5μl
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|-
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|dNTPs
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|1μl
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|-
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|Primer 1
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|1μl
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|-
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|Primer 2
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|1μl
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|-
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|bacteria
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|(about 1µl = 1 colony)
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|-
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|Dream taq
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|0.5μl
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|}
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Primer used:
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For cloning in Topo vector = Pu Pr (universal primer)
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For pPS2 = iPS 66/67
{| class="wikitable centre" width="80%"
{| class="wikitable centre" width="80%"
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|-
| width="25%" |
| width="25%" |
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Initial denaturation
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Bacteria lysis
| width="25%" |
| width="25%" |
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94°C
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95°C
| width="25%" |
| width="25%" |
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1 min
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5 min
| width="25%" |
| width="25%" |
1
1
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| 1
| 1
|}
|}
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[[File:0809 PCR clones.jpg|400px|center]]
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We identify that we have success with to clone of PS. (well 5-6)
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====Liquide culture of clones====
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The PS clone that reveal to be ok are cultivate in LB + AMP médium
 +
 +
====Checking PCR====
 +
Electrophoresis of the PCR made [http://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#PCR_LS_PS_GS_Cad Friday]
 +
 +
LS PS GS CAD (and chromo) (2 enzymes = vent taq and dream taq)
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[[File:0809 PCR LSPSGSCADCRHOMO vent dream.jpg|400px|center]]
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====Cloning====
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With this PCR, I do a cloning in a Topo Cloning vector (Zero Blunt TOPO PCR Cloning Kit) (LS GS and CAD)
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 +
{| class="wikitable centre" width="50%"
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|+
 +
|-
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! scope=col | component
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! scope=col | volume
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|-
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|PCR product
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|1μl
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|-
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|Salt
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|1μl
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|-
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|Vector
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|1 μl
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|-
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|H<sub>2</sub>O
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|3μl
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|}
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Between 5 and 30 min at room temperature
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==== Transformation====
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Add 50µL of bacteria to the clonage previously done
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30min on ice
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45 sec at 42°C
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add 1ml of LB
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1hour at 37°
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Spread on a petri dish
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===Construction of the fusion protein===
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We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6
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==Photo of the Day==
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[[File:Paris Saclay 8_september.jpg|150px|center]]
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[http://ifris.org/membre/morgan-meyer/ Morgan Meyer] , one of those few we have interviewed during this month of september.
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To learn more about the ethic aspect of our project, just click [http://2014.igem.org/Team:Paris_Saclay/Ethics here]
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 +
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{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 12:55, 14 October 2014

Contents

Monday 8th September

Lab Work

by Mélanie

Lemon Scent

PCR with bacteria

From the petri dish (made here, I select some clone to do a PCR : clones 1-2-3 for cad clones 4-5 for PS clones 6 for LS

component volume
H2O 41.5μl
buffer 5μl
dNTPs 1μl
Primer 1 1μl
Primer 2 1μl
bacteria (about 1µl = 1 colony)
Dream taq 0.5μl

Primer used:

For cloning in Topo vector = Pu Pr (universal primer)

For pPS2 = iPS 66/67

Cycle step Temperature Time Cycle

Bacteria lysis

95°C

5 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
0809 PCR clones.jpg

We identify that we have success with to clone of PS. (well 5-6)

Liquide culture of clones

The PS clone that reveal to be ok are cultivate in LB + AMP médium

Checking PCR

Electrophoresis of the PCR made Friday

LS PS GS CAD (and chromo) (2 enzymes = vent taq and dream taq)

0809 PCR LSPSGSCADCRHOMO vent dream.jpg

Cloning

With this PCR, I do a cloning in a Topo Cloning vector (Zero Blunt TOPO PCR Cloning Kit) (LS GS and CAD)

component volume
PCR product 1μl
Salt 1μl
Vector 1 μl
H2O 3μl


Between 5 and 30 min at room temperature

Transformation

Add 50µL of bacteria to the clonage previously done

30min on ice 45 sec at 42°C

add 1ml of LB

1hour at 37°

Spread on a petri dish

Construction of the fusion protein

We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6

Photo of the Day

Paris Saclay 8 september.jpg

Morgan Meyer , one of those few we have interviewed during this month of september.

To learn more about the ethic aspect of our project, just click here


Back to the calendar