IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/06

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Raphaël, Théotime and Stéphane

Miniprep - Kit Qiagen - Final volume : 50µL (Raphaël)

  • pSUlib - attC KanR

Miniprep - Kit Promega (Stéphane)

  • pSUlib - attC KanR

Restriction digest - Xba I

  • pSUlib - attC KanR (Qiagen and Promega Minipreps)

Final volume : 20 µL
15 µL distilled water
2 µL minipreped DNA
0,2 µL BSA 100x
2 µL buffer 10x
1 µL restriction enzyme

Protocol
1- Add in order listed above.
2- Vortex the mix 2 sec, spindown in micro-centrifuge and put in 37°C for 2h.

DNA Gel electrophoresis (agarose 1,5% w/v)
7,5g of agarose in 500mL of TBE buffer 0,5x
2 µL of EtBr was added in the gel

Protocol (2 wells)

  • 10 µL of minipreped DNA (Qiagen) + 2 µL of loading buffer 6x
  • 10 µL of ladder 1Kb

Migration : 50V, 1h30

DNA Gel electrophoresis (agarose 1,0% w/v)
2 µL of EtBr was added in the gel

Protocol (2 wells)

  • 10 µL of minipreped DNA (Promega) + 2 µL of loading buffer 6x
  • 10 µL of ladder 1Kb

Migration : 50V, 1h30

Image:electrophoresis_060710.jpg
The bands of the ladder are not well separated. Thus, the migration should last longer and the gel should be at 1,0% agarose.


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