Team:Paris Saclay/Notebook/September/8

From 2014.igem.org

(Difference between revisions)
(Checking PCR)
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[photo]
[photo]
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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)
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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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|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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===B-Contruction of the fusion protein===
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We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6
====PCR with bacteria====
====PCR with bacteria====

Revision as of 15:54, 9 September 2014

Contents

Lab Work

D-Lemon Scent

Checking PCR

Electrophoresis of the PCR made Friday

[photo]

Cloning

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

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

B-Contruction of the fusion protein

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

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

[photo]

We identify that we have success with to clone of PS.

Liquide culture of clones

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