Team:Paris Liliane Bettencourt/Notebook/2010/07/06/
From 2010.igem.org
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
<a href="" class="image" title="Image:electrophoresis_060710.jpg"><img alt="Image:electrophoresis_060710.jpg" src="
" width="621" height="433" border="0" /></a>
The bands of the ladder are not well separated. Thus, the migration should last longer and the gel should be at 1,0% agarose.