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Contents

Extraction of EnvZ* and OmpR* from E. coli genome

Name of the PCR

Name What's in ? Template DNA Oligo F Oligo R
PCR 147 EnvZ* S 120 O 126 O 127
PCR 148 OmpR* S 119 O 138 O 139
T 147 nothing no template O 126 O 127
T 148 nothing no template O 138 O 139

Protocol

Protocol

  • 10 µL Phusion HF Buffer 5X
  • 2.5 µL Oligo F 10 mM
  • 2.5 µL Oligo R 10 mM
  • 1 µL dNTP
  • 1 µL Template DNA
  • 33 µL H2O

PCR Programme

  • 98°C 30s Initial denaturation
  • CYCLE 30X
  • 98°C 10s Denaturation
  • 60°C 30s Annealing
  • 72°C 45s Elongation
  • END OF CYCLE
  • 72°C 5min Terminal Elongation

Resuts

Electrophoresis settings

  • Gel 1% agar
  • 10µL Ladder 1kb
  • 10µL Ladder 100bp
  • 4µL DNA + 2µL Loading Blue

Results of the electrophoresis

Name Gene Well Expected size Measured size
PCR 147 EnvZ* 2 1412 bp
T 147 nothing 3 nothing
PCR 148 OmpR* 4 779 bp
T 148 nothing 5 nothing

Cleaning of the PCR products

Digestion of the PCR products

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
  • 5 hours at room temperature
  • transformation of TOP10 competent cells with 5 µL of ligation product
  • spreading on LB plates + ampicilline and incubation overnight at 37°C