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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
PCR3
- PCRFliA1 (O143-F / O152-R)
- PCRFliA2 (O153-F / O150-R)
- PCRFliA3 (O148-F / O150-R)
Program: PCRFliA1
Denaturation :
98°C-5'
Cycling 1 (30X) :
98°C-10"
Gradient 66°C +/-6°C - 25
72°C-20"
Elongation :
72°C-5'
Program: PCRFliA2
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
Gradient 61°C +/-10°C - 25
72°C-20"
Cycling 2 (30X) :
98°C-10"
72°C-30"
Elongation :
72°C-5'
Program: PCRFliA3
Denaturation :
98°C-30'
Cycling 1 (2X) :
98°C-10"
72°C-30"
Cycling 2 (5X) :
98°C-10"
98°C->72°C low decreasing 1.0°C/s
72°C-30"
Elongation :
72°C-5'
| Name
| Promotor
| Well
| Expected size
| Measured size
|
| PCRFliA1
| Upstream part of FliA
| 2-27
| 197 bp
| ~ 200 bp
|
| PCRFliA2
| Downstream part of FliA
| 28-54
| 0 bp
| 0 bp
|
| PCR
| pFliA+rbs
| 55-56
| 325 bp
| 0 bp
|
| PCR
| PFliA
| 57-58
| 310 bp
| 0 bp
|
| PCR
| pFliA+Gene FliA
| 59-60
| 1100 bp
| 0 bp
|
Cloning of EnvZ*
Concentration measurement by Biophotometer
| Name
| Digestion n°
| [DNA] in µg/mL
| A260/A280
|
| EnvZ*
| D159
| 10 µg/mL
| 1.35
|
| pSB1A2
| D116
| 17 µg/mL
| 1.02
|
Ligation
|
| control
| insert / vector ratio
|
|
|
| 1,8 / 1
| 2,4 / 1
| 3,1 / 1
|
| D159 EnvZ* (µL)
| 0
| 6
| 8
| 8
|
| D116 pSB1A2 (µL)
| 2
| 2
| 2
| 1,5
|
| 10X T4 ligase (µL)
| 2
| 2
| 2
| 2
|
| T4 DNA ligase (µL)
| 1
| 1
| 1
| 1
|
| water (µL)
| 15
| 9
| 7
| 7,5
|
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