User:DavidC/24 September 2009
From 2009.igem.org
Thursday the 24th
Restriction digest
Ligation between BBa_P1003 and BBa_B0014
Restriction digest of BBa_P1003 (3675bp) by Spe and Pst:
DNA (10µg final) = 4µL
Buffer H (TAKARA) =2µL
H20 = 12µL
Spe1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
>
Incubation 1h at 37°C.
Restriction digest of BBa_B0014 (95bp) by XbaI and PstI:
DNA (10µg final) = 4µL
Buffer M (TAKARA) =2µL
H20 = 12µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
>
Incubation 1h at 37°C.
Ligation between BBa_C0012 and BBa_B0014
Restriction digest of BBa_C0012 (3207bp) by PstI and SpeI:
DNA (10µg final) = 6,47µL
Buffer H (TAKARA) =2µL
H20 = 9,63µL
Spe1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
>
Incubation 1h at 37°C.
Restriction digest of BBa_BB014 (95bp) by XbaI and PstI:
DNA (10µg final) = 4,16µL
Buffer H (TAKARA) =2µL
H20 = 11,84µL
Xba1 (TAKARA) = 1µL
Pst1 (TAKARA) = 1µL
>
Incubation 1h at 37°C.
Ligation between BBa_B0030 and BBa_B0014 + BBa_J23100
Ligation between BBa_R0010 and BBa_B0014
Ligation between BBa_I712612 and BBa_B0014
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples:
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.