Team:Paris/July 24

MiniPreps

 * The Promega MiniPreps protocol has been used on all the clones cultivated on the 23th.

Digestion Mix
10µl of Miniprep (26 aug.) 12.5µl of water 2.5µl of Buffer N°2 0.25µl of BSA 100x 1µl of enzyme 1 1µl of enzyme 2


 * Incubation 1h at 37°C with the first enzyme
 * Add the second enzyme
 * Incubation 1h at 37°C with the second enzyme
 * Store on ice
 * Revelation of the digestion by electrophoresis on agarose gel

Results of digestions : Electrophoresis
conditions :
 * 10µl of ladder 1 kb (except for gel n°7 : 100 pb)
 * 30µl of digestion added with 5µl of loading Dye 6x
 * migration ~30min at 100W
 * Gel 1, 2, 3, 4, 5, 6, 8 = 0.8%
 * Gel 7 = 1,2%

gel1 gel2 gel3 gel4

gel5 gel6 gel7 gel8

==> Conclusion: Most of the digestion have succeed

Extraction of the DNA

 * Cutting of the parts of interest, for all the digestion that have migrated on the gels
 * Store of all the piece of gel O/N at -20°C.