Team:Paris Saclay/Notebook/September/8
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+ | {{Team:Paris_Saclay/notebook_header}} | ||
+ | =Monday 8th September= | ||
+ | |||
==Lab Work== | ==Lab Work== | ||
+ | ''by Mélanie'' | ||
- | === | + | ===Lemon Scent=== |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
====PCR with bacteria==== | ====PCR with bacteria==== | ||
From the petri dish ([https://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#Transformation made here], I select some clone to do a PCR : | From the petri dish ([https://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#Transformation made here], I select some clone to do a PCR : | ||
Line 12: | Line 11: | ||
clones 4-5 for PS | clones 4-5 for PS | ||
clones 6 for LS | clones 6 for LS | ||
+ | |||
+ | {| class="wikitable centre" width="50%" | ||
+ | |+ | ||
+ | |- | ||
+ | ! scope=col | component | ||
+ | ! scope=col | volume | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |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 | ||
{| class="wikitable centre" width="80%" | {| class="wikitable centre" width="80%" | ||
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| 1 | | 1 | ||
|} | |} | ||
+ | |||
+ | [[File:0809 PCR clones.jpg|400px|center]] | ||
+ | |||
+ | 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 [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/5#PCR_LS_PS_GS_Cad Friday] | ||
+ | |||
+ | LS PS GS CAD (and chromo) (2 enzymes = vent taq and dream taq) | ||
+ | |||
+ | [[File:0809 PCR LSPSGSCADCRHOMO vent dream.jpg|400px|center]] | ||
+ | |||
+ | ====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 | ||
+ | |||
+ | ===Construction of the fusion protein=== | ||
+ | We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6 | ||
+ | |||
+ | ==Photo of the Day== | ||
+ | [[File:Paris Saclay 8_september.jpg|150px|center]] | ||
+ | |||
+ | [http://ifris.org/membre/morgan-meyer/ Morgan Meyer] , one of those few we have interviewed during this month of september. | ||
+ | |||
+ | To learn more about the ethic aspect of our project, just click [https://2014.igem.org/Team:Paris_Saclay/Ethics here] | ||
+ | |||
+ | |||
+ | {{Team:Paris_Saclay/notebook_footer}} |
Latest revision as of 12:55, 14 October 2014
Contents |
Monday 8th September
Lab Work
by Mélanie
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
Construction of the fusion protein
We check the sequence of the fusion protein and identify a clone with our chromoprotein = W6
Photo of the Day
Morgan Meyer , one of those few we have interviewed during this month of september.
To learn more about the ethic aspect of our project, just click here