User:DavidC/28 September 2009
From 2009.igem.org
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Monday the 28th
DNA extraction
DNA extraction with miniprep (promega).
Samples:
BBa_P1003 + BBa_B0014;
BBa_C0012 + BBa_B0014;
BBa_B0014 + BBa_J23100 + BBa_B0030;
BBa_R0010 + BBa_B0014.
BBa_I712669 + BBa_B0014
Final volume = 30µL
Restriction digest
Restriction digest of I712669+B0014 by EcoRI and SpeI (4806bp):
DNA (miniprep) = 30µL
Buffer 2 (NEB) =5µL
H20 = 13µL
Eco RI (NEB) = 1µL
Spe I (NEB) = 1µL
1 hour of incubation at 37°C.
Restriction digest of P1003 by EcoRI and XbaI:
DNA (miniprep) = 30µL
Buffer 2 (NEB) =5µL
H20 = 13µL
Eco RI (NEB) = 1µL
Xba I (NEB) = 1µL
1 hour of incubation at 37°C.
Restriction digest of C0012 + B0014 by XbaI and PstI:
DNA (miniprep) = 30µL
Buffer 2 (NEB) =5µL
H20 = 13µL
Xba 1 (NEB) = 1µL
Pst 1 (NEB) = 1µL
1 hour of incubation at 37°C.
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples: BBa_P1003, BBa_I712669 + BBa_B0014, BBa_C0012 + BBa_B0014.
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.