User:DavidC/10 September 2009

Restriction digest of C0012 (concentration = 1,49µg/µL) by EcoRI and SpeI (1128bp):
DNA (10µg final) = 6,7µL Buffer Eco R1 (NEB) = 5µL H20 = 35,8µL BSA = 0,5µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

Ligation between R0040 and B0030:
Restriction digest of B0030 (=0,245µg/µL) by EcoRI and XbaI (2094bp):

DNA (10µg final) = 40,8µL Buffer Eco R1 (NEB) = 5µL H20 = 3,2µL Eco R1 = 1µL Incubation 1h at 37°C.

DNA purification with a nucleospin (macherey-nagel).

DNA = 50µL Buffer 2 (NEB) = 6µL BSA (NEB) = 0,6µL H20 = 2,4µL Xba 1 = 1µL Incubation 1h at 37°C.

Restriction digest of R0040 (1,75µg/µL) by EcoRI and SpeI (54bp):

DNA (10µg final) = 5,7µL Buffer Eco R1 (NEB) = 5µL H20 = 36,3µL BSA = 0,5µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

DNA electrophoresis
85 Volt, 15 minutes. 105 Volt, 40 minutes. Ladder fermentas 1 Kb.



Samples: BBa_B0030, BBa_R0040, BBa_C0012.

DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.