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User:DavidC/13 September 2009
From 2009.igem.org
Contents |
Sunday the 13th
Restriction
Ligation of C0012 with B0014:
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.
TAKARA Enzyme testing
Ligation of C0012 with B0014:
B0014 (= 2,405µg/µL) restriction digest with EcoRI and XbaI (3284bp):
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.
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.
