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Team:Paris Saclay/Notebook/September/8
From 2014.igem.org
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 |
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)
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
