User:DavidC/15 September 2009

Ligation between BBa_I712669 and BBa_B0014:
I712669 (= 6,667µg/µL) restriction digest with PstI and XbaI (1380bp) :

DNA (10µg final) = 1,5µL Buffer 3 (NEB) = 5µL H20 = 41,5µL Pst 1 = 1µL Xba 1 = 1µL Incubation 1h at 37°C.

BBa_B0014 (= 2,381µg/µL) restriction digest by PstI and SpeI (3284bp):

DNA (10µg final) = 4,2µL Buffer 2 (NEB) = 5µL H20 = 38,8µL Pst 1 = 1µL Spe 1 = 1µL Incubation 1h at 37°C.

Ligation between BBa_B0014 and BBa_P1003
BBa_P1003 (= 2,17µg/µL) restriction digest by PstI and SpeI (3675bp):

DNA (10µg final) = 2,4µL Buffer 3 (NEB) = 5µL H20 = 40,6µL Pst 1 = 1µL Spe1 = 1µL Incubation 1h at 37°C.

BBa_B0014 (=2,38µg/µL) by PstI and XbaI (95bp):

DNA (10µg final) = 4,2µL Buffer 2 (NEB) = 5µL H20 = 38,8µL Pst1 = 1µL Xba1 = 1µL Incubation 1h at 37°C.

Ligation between BBa_J23100 with BBa_B0014
BBa_J23100 (=1,67µg/µL) restriction digest by EcoRI and XbaI (35bp): DNA (10µg final) = 6µL Buffer M (TAKARA) = 5µL H20 = 7µL EcoRI (TAKARA) = 1µL Xba 1 (TAKARA) = 1µL Incubation 1h at 37°C.

BBa_B0014 (= 2,81µg/µL) restriction digest by PstI and SpeI (3284bp):

DNA (10µg final) = 4,2µL Buffer H (TAKARA) = 5µL H20 = 38,8µL Pst 1 (TAKARA) = 1µL Spe 1 (TAKARA) = 1µL Incubation 1h at 37°C.

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

Samples: BBa_J23100, BBa_B0014, BBa_I712669, BBa_P1003, BBa_B0014, BBa_B0014.



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