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User:DavidC/13 September 2009
From 2009.igem.org
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=== Sunday the 13th === | === Sunday the 13th === | ||
| - | ==== Restriction ==== | + | ==== Restriction digest ==== |
| - | ==== Ligation | + | ==== Ligation between BBa_C0012 and BBa_B0014: ==== |
| - | + | ||
| + | BBa_B0014 (=2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp): <br> | ||
DNA (10µg final)= 4,2µL <br> | DNA (10µg final)= 4,2µL <br> | ||
| Line 25: | Line 27: | ||
Eco R1 = 1µL <br> | Eco R1 = 1µL <br> | ||
Incubation 1h at 37°C. <br> | Incubation 1h at 37°C. <br> | ||
| + | |||
DNA purification with a nucleospin (macherey-nagel) . <br> | DNA purification with a nucleospin (macherey-nagel) . <br> | ||
| + | |||
DNA = 50µL <br> | DNA = 50µL <br> | ||
Buffer 2 (NEB) = 6µL <br> | Buffer 2 (NEB) = 6µL <br> | ||
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H20 = 2,4µL <br> | H20 = 2,4µL <br> | ||
Xba 1 = 1µL <br> | Xba 1 = 1µL <br> | ||
| - | |||
Incubation 1h at 37°C. <br><br> | Incubation 1h at 37°C. <br><br> | ||
C0012 (1,55µg/µL) restriction digest by EcoRI and SpeI (1128bp): <br> | C0012 (1,55µg/µL) restriction digest by EcoRI and SpeI (1128bp): <br> | ||
| + | |||
DNA (10µg final) = 6,4µL <br> | DNA (10µg final) = 6,4µL <br> | ||
Buffer Eco R1 (NEB) = 5µL <br> | Buffer Eco R1 (NEB) = 5µL <br> | ||
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BSA = 0,5µL <br> | BSA = 0,5µL <br> | ||
Eco R1 (NEB) = 1µL <br> | Eco R1 (NEB) = 1µL <br> | ||
| - | |||
Spe 1 (NEB) = 1µL <br> | Spe 1 (NEB) = 1µL <br> | ||
| - | |||
Incubation 1h at 37°C. <br> | Incubation 1h at 37°C. <br> | ||
==== TAKARA Enzyme testing ==== | ==== TAKARA Enzyme testing ==== | ||
| - | ==== Ligation | + | ==== Ligation between BBa_C0012 and BBa_B0014: ==== |
| - | B0014 (= 2,405µg/µL) restriction digest with EcoRI and XbaI ( | + | |
| + | B0014 (= 2,405µg/µL) restriction digest with EcoRI and XbaI (3128bp): <br> | ||
| + | |||
ADN (10µg final) = 4,2µL <br> | ADN (10µg final) = 4,2µL <br> | ||
Buffer M (TAKARA) = 5µL <br> | Buffer M (TAKARA) = 5µL <br> | ||
| Line 54: | Line 58: | ||
Eco R1 = 1µL <br> | Eco R1 = 1µL <br> | ||
Xba 1 = 1µL <br> | Xba 1 = 1µL <br> | ||
| - | |||
Incubation 1h à 37°C. <br> | Incubation 1h à 37°C. <br> | ||
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Ladder fermentas 1 Kb. <br> | Ladder fermentas 1 Kb. <br> | ||
| - | Samples: BBa_B0014, | + | Samples: BBa_B0014, BBa_C0013, BBa_B0014. |
| - | [[ | + | [[image:SU1309.png|center]] |
==== DNA purification ==== | ==== DNA purification ==== | ||
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br> | Kit Qiagen “gel extraction kit”, final volume = 50µL. <br> | ||
Latest revision as of 14:37, 21 October 2009
Introduction Current therapies Gene therapies DVS Concept Tissue Vector Cell Vector Therapeutic plasmid Conclusion Implementation Tissue targeting Antitumor action Treatment modeling Conclusion
BioBricks Material & Method Bibliography
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.
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.
