User:DavidC/10 September 2009
From 2009.igem.org
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Thursday the 10th
Restriction digest
Restriction digest of C0012 (concentration = 1,49µg/µL) by EcoRI and SpeI (1128bp):
DNA (10µg final) = 6,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 35,8µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
Ligation between R0040 and B0030:
Restriction digest of B0030 (=0,245µg/µL) by EcoRI and XbaI (2094bp):
DNA (10µg final) = 40,8µL
Buffer Eco R1 (NEB) = 5µL
H20 = 3,2µL
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.
Restriction digest of R0040 (1,75µg/µL) by EcoRI and SpeI (54bp):
DNA (10µg final) = 5,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 36,3µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples:
BBa_B0030, BBa_R0040, BBa_C0012.
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.