Team:Paris Saclay/Notebook/September/8
From 2014.igem.org
(→Checking PCR) |
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[photo] | [photo] | ||
+ | |||
+ | ====Cloning==== | ||
+ | With this PCR, I do a cloning in a Topo Cloning vector (Zero Blunt TOPO PCR Cloning Kit) | ||
+ | |||
+ | {| 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=== | ||
+ | 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