From 2008.igem.org
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| Phusion=0,5µL<br> | | Phusion=0,5µL<br> |
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- | =PCR3=
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- | * PCRFliA1 (O143-F / O152-R)
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- | * PCRFliA2 (O153-F / O150-R)
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- | * PCRFliA3 (O148-F / O150-R)
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- | ----
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- | '''Program: PCRFliA1'''<br>
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- | '''''Denaturation :''''' <br>
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- | 98°C-5'<br>
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- | '''''Cycling 1 (30X) :''''' <br>
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- | 98°C-10"<br>
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- | Gradient 66°C +/-6°C - 25''<br>
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- | 72°C-20"<br>
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- | '''''Elongation :''''' <br>
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- | 72°C-5'<br>
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- | '''Program: PCRFliA2'''<br>
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- | '''''Denaturation :''''' <br>
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- | 98°C-5'<br>
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- | '''''Cycling 1 (3X) :''''' <br>
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- | 98°C-10"<br>
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- | Gradient 61°C +/-10°C - 25''<br>
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- | 72°C-20"<br>
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- | '''''Cycling 2 (30X) :''''' <br>
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- | 98°C-10"<br>
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- | 72°C-30"<br>
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- | '''''Elongation :''''' <br>
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- | 72°C-5'<br>
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- | '''Program: PCRFliA3'''<br>
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- | '''''Denaturation :''''' <br>
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- | 98°C-30'<br>
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- | '''''Cycling 1 (2X) :''''' <br>
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- | 98°C-10"<br>
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- | 72°C-30"<br>
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- | '''''Cycling 2 (5X) :''''' <br>
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- | 98°C-10"<br>
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- | 98°C->72°C low decreasing 1.0°C/s<br>
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- | 72°C-30"<br>
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- | '''''Elongation :''''' <br>
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- | 72°C-5'<br>
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| =Cloning of EnvZ*= | | =Cloning of EnvZ*= |
Revision as of 11:18, 26 August 2008
← Yesterday ↓ Calendar ↑Tomorrow →
PCR
PCR1
- pFlhDC (O111-F / O113-R)
- Gene FlhDC (O134-F / O131-R)
- pFlgB (O102-F / O103-R)
- pFlhB (O108-F / O109-R)
Program: Gradient
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
Gradient 61°C +/-10°C - 30
72°C-30"
Cycling 2 (25X) :
98°C-10"
72°C-30"
Elongation :
72°C-5'
Vf=20µL
H20=13,4µL
Buffer5X=4µL
dNTP=0,4µL
O1=1µL
O2=1µL
Phusion=0,2µL
PCR2
- pFliA (rbs) (O145-F / O144-R)
- pFliA (O145-F / O146-R)
- pFliA +Gene FliA (O145-F / O151-R)
Program: promoter
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
55°C - 30
72°C-30"
Cycling 2 (30X) :
98°C-10"
65°C-30"
72°C-30"
Elongation :
72°C-5'
Vf=50µL
H20=33,5µL
Buffer5X=10µL
dNTP=1µL
O1=2,5µL
O2=2,5µL
Phusion=0,5µL
Cloning of EnvZ*
Concentration measurement by Biophotometer
Name
| [DNA] in µg/mL
| A260/A280
|
EnvZ*
| 10 µg/mL
| 1.35
|
pSB1A2
| 17 µg/mL
| 1.02
|
Ligation
|