Team:Paris/August 9

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Contents

Ligation Day

List of ligations

Ligation name Insert name Vector name
L 132 D 139 (flhDC (gene)) D 142 (pSB1A2)
L 133 D 140 (OmpR*) D 142 (pSB1A2)
L 134 D 141 (EnvZ*) D 142 (pSB1A2)
L 135 D 133 (pflgA) D 137 (pSB3K3)
L 136 D 134 (pflgB) D 137 (pSB3K3)
L 137 D 135 (pflhB) D 137 (pSB3K3)
L 138 D 138 (E0240) D 137 (pSB3K3)


Measure of DNA concentration

First of all, we have to determine the concentration of DNA of the different templates.

We used the biophotometer.

  • Sample: 5µL of template DNA + 95µL of water
  • Blank: 5µL of EB buffer + 95 µL of water
Ligation name [DNA] diluted
ng/µL
[DNA] template
ng/µL
D 132 1 20
D 133 1 20
D 134 1 20
D 137 1 20
D 138 1 20
D 139 0 0
D 140 1 20
D 141 2 40
D 142 1 20

Protocol of the ligation

  • 2 µL T4 Ligase Buffer 10X
  • X µg/µL vector
  • 3 or 4 x X µg/µL insert
  • Pure water qsp 20 µL
  • 1 µL T4 ligase
  • O/N at 16°C + sunday 16°C
Ligation name Insert Volume Vector Volume
L 132 3 µL 5 µL
L 133 3 µL 5 µL
L 134 3 µL 5 µL
L 135 2.5 µL 2.5 µL
L 136 2.5 µL 2.5 µL
L 137 2.5 µL 2.5 µL
L 138 2.5 µL 2.5 µL