Team:Paris Saclay/Notebook/September/8

From 2014.igem.org

(Difference between revisions)
(Cloning)
(Lab Work)
Line 2: Line 2:
===D-Lemon Scent===
===D-Lemon Scent===
-
====Checking PCR====
 
-
Electrophoresis of the PCR made [http://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#PCR_LS_PS_GS_Cad 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)
 
-
 
-
{| class="wikitable centre" width="50%"
 
-
|+
 
-
|-
 
-
! scope=col | component
 
-
! scope=col | volume
 
-
|-
 
-
|PCR product
 
-
|1μl
 
-
|-
 
-
|Salt
 
-
|1μl
 
-
|-
 
-
|Vector
 
-
|1 μl
 
-
|-
 
-
|H<sub>2</sub>O
 
-
|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====
====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 :
Line 132: Line 88:
We identify that we have success with to clone of PS.
We identify that we have success with to clone of PS.
 +
====Liquide culture of clones====
====Liquide culture of clones====
The PS clone that reveal to be ok are cultivate in LB + AMP médium
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]
 +
 +
[photo]
 +
 +
====Cloning====
 +
With this PCR, I do a cloning in a Topo Cloning vector (Zero Blunt TOPO PCR Cloning Kit) (LS GS and CAD)
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | component
 +
! scope=col | volume
 +
|-
 +
|PCR product
 +
|1μl
 +
|-
 +
|Salt
 +
|1μl
 +
|-
 +
|Vector
 +
|1 μl
 +
|-
 +
|H<sub>2</sub>O
 +
|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===
===B-Contruction of the fusion protein===
We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6
We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6

Revision as of 12:26, 14 September 2014

Contents

Lab Work

D-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

[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

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

B-Contruction of the fusion protein

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