User:DavidC/24 September 2009

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
Restriction digest of BBa_B0030 (2094bp) by XbaI and EcoRI:

DNA (10µg final) = 10,25µL Buffer M (TAKARA) =2µL H20 = 2,75µL EcoR1 (TAKARA) = 1µL Xba1 (TAKARA) = 1µL > Incubation 1h at 37°C.

Restriction digest of BBa_J23100 + BBa_B0014 by SpeI and EcoRI:

DNA (10µg final) = 2,57µL Buffer M (TAKARA) =2µL H20 = 13,43µL EcoR1 (TAKARA) = 1µL Spe1 (TAKARA) = 1µL > Incubation 1h at 37°C.

Ligation between BBa_R0010 and BBa_B0014
Restriction digest of BBa_R0010 (2279bp) by XbaI and EcoRI:

DNA (10µg final) = 55,6µL Buffer H (TAKARA) =6µL H20 = 5,4µL EcoR1 (TAKARA) = 1µL Xba1 (TAKARA) = 1µL > Incubation 1h at 37°C.

Restriction digest of BBa_B0014 (95bp) by SpeI and EcoRI:

DNA (10µg final) = 4,16µL Buffer H (TAKARA) =2µL H20 = 11,84µL EcoR1 (TAKARA) = 1µL Spe 1 (TAKARA) = 1µL > Incubation 1h at 37°C.

Ligation between BBa_I712669 and BBa_B0014
Restriction of BBa_I712669 (4806bp) by SpeI and PstI:

DNA (10µg final) = 4µL Buffer H (TAKARA) =2µL H20 = 12µL Pst1 (TAKARA) = 1µL Spe 1 (TAKARA) = 1µL > Incubation 1h at 37°C.

Restriction digest of B0014 (95bp) by XbaI and PstI:

Same sample as the ligation between BBa_C0012 and BBa_B0014.

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

Samples: BBa_C0012, BBa_BB014 (XbaI, PstI), BBa_B0030, BBa_J23100 + BBa_B0014, BBa_R0010, BBa_B0014 (SpeI, EcoRI), BBa_I712669.

We have not the picture (technical problem).

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