Team:Paris Saclay/Notebook/September/8

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(Lab Work)
(Cloning)
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====Cloning====
====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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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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Revision as of 12:25, 14 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) (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


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

B-Contruction of the fusion protein

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