User:DavidC/13 September 2009

From 2009.igem.org

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Contents

Sunday the 13th

Restriction digest

Ligation between BBa_C0012 and BBa_B0014:

BBa_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 between BBa_C0012 and BBa_B0014:

B0014 (= 2,405µg/µL) restriction digest with EcoRI and XbaI (3128bp):

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.

Samples: BBa_B0014, BBa_C0013, BBa_B0014.

SU1309.png

DNA purification

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