User:DavidC/20 September 2009
From 2009.igem.org
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Sunday the 20th
Restriction digest
P1003 (= 2,5µg/µL) restriction digest by EcoRI and SpeI:
DNA (10µg final) = 4µL
Buffer H (TAKARA) =2µL
H20 = 12µL
EcoR1 (TAKARA) = 1µL
Spe 1 (TAKARA) = 1µL
>
Incubation 1h at 37°C.
Calf intestine phosphatase (CIP) processing:
Restriction digest mix = 20µL
CIP Buffer (TAKARA) = 5µL
H20 = 24µL
CIP (TAKARA) = 1µL
1h at 37°C
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
First: Purification of P1003 fragment:
Second: Quantization of P1003; C0012 and B0014:
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.