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