User:DavidC/13 September 2009

Ligation between BBa_C0012 and BBa_B0014:
BBa_B0014 (=2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp):

DNA (10µg final)= 4,2µL Buffer Eco R1 (NEB) = 5µL H20 = 39,8µL 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.

C0012 (1,55µg/µL) restriction digest by EcoRI and SpeI (1128bp):

DNA (10µg final) = 6,4µL Buffer Eco R1 (NEB) = 5µL H20 = 35,6µL BSA = 0,5µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

Ligation between BBa_C0012 and BBa_B0014:
B0014 (= 2,405µg/µL) restriction digest with EcoRI and XbaI (3128bp):

ADN (10µg final) = 4,2µL Buffer M (TAKARA) = 5µL H20 = 38,8µL Eco R1 = 1µL Xba 1 = 1µL Incubation 1h à 37°C.

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

Samples: BBa_B0014, BBa_C0013, BBa_B0014.



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