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| - |
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| - |
| |
| - | === Friday the 18th ===
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| - |
| |
| - | ==== Restriction digest ====
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| - |
| |
| - | ==== Ligation between B0014 and P1003 ====
| |
| - |
| |
| - | Restriction digest of BBa_B0014 (= 2,41µg/µL) by EcoRI and XbaI (3284bp): <br>
| |
| - |
| |
| - | DNA (10µg final) = 4,2µL <br>
| |
| - | Buffer Eco R1 (NEB) = 5µL <br>
| |
| - | H20 = 39,8µL <br>
| |
| - | Eco R1 = 1µL <br>
| |
| - | Incubation 1h at 37°C. <br>
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| - |
| |
| - | DNA purification with a nucleospin (macherey-nagel). <br>
| |
| - |
| |
| - | DNA = 50µL <br>
| |
| - | Buffer 2 (NEB) = 6µL <br>
| |
| - | BSA (NEB) = 0,6µL <br>
| |
| - | H20 = 2,4µL <br>
| |
| - | Xba 1 = 1µL <br>
| |
| - | Incubation 1h at 37°C. <br><br>
| |
| - |
| |
| - | Restriction digest of P1003 (4,17µg/µL) by EcoRI and SpeI (967bp): <br>
| |
| - |
| |
| - | DNA (10µg final) = 2,4µL <br>
| |
| - | Buffer Eco R1 (NEB) = 5µL <br>
| |
| - | H20 = 40,6µL <br>
| |
| - | BSA = 0,5µL <br>
| |
| - | Eco R1 (NEB) = 1µL <br>
| |
| - | Spe 1 (NEB) = 1µL <br>
| |
| - | Incubation 1h at 37°C. <br>
| |
| - |
| |
| - | ==== Ligation of B0014 with C0012 ====
| |
| - |
| |
| - | B0014 (= 2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp): <br>
| |
| - |
| |
| - | Same samples as the restriction digest used for B0014 and P1003 ligation. <br><br>
| |
| - |
| |
| - | C0012 (1,56µg/µL) restriction digest by EcoRI and SpeI (1128bp): <br>
| |
| - |
| |
| - | DNA (10µg final) = 6,4µL <br>
| |
| - | Buffer Eco R1 (NEB) = 5µL <br>
| |
| - | H20 = 36,1µL <br>
| |
| - | BSA = 0,5µL <br>
| |
| - | Eco R1 (NEB) = 1µL <br>
| |
| - | Spe 1 (NEB) = 1µL <br>
| |
| - | Incubation 1h at 37°C. <br>
| |
| - |
| |
| - | ==== DNA electrophoresis ====
| |
| - |
| |
| - | 85 Volt, 15 minutes. <br>
| |
| - | 105 Volt, 40 minutes. <br>
| |
| - | Ladder fermentas 1 Kb. <br>
| |
| - |
| |
| - |
| |
| - | Samples:
| |
| - |
| |
| - | ==== DNA purification ====
| |
| - |
| |
| - | Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
| |
| - |
| |
| - | ==== Ligation ====
| |
| - |
| |
| - | Ligation schemes: plasmid / insert: <br>
| |
| - |
| |
| - | BBa_B0014/BBa_P1003 ; <br>
| |
| - | BBa_B0014/BBa_C0012. <br>
| |
| - |
| |
| - | First report: <br>
| |
| - | Plasmid = 1µL <br>
| |
| - | Insert = 5µL <br>
| |
| - |
| |
| - | Second report: <br>
| |
| - | Plasmid = 1µL <br>
| |
| - | Insert = 3µL <br>
| |
| - |
| |
| - | Third report: <br>
| |
| - | Plasmid = 1µL <br>
| |
| - | Insert = 7µL <br>
| |
| - |
| |
| - | ==== NEB Enzymes ====
| |
| - |
| |
| - | For each samples add sterilized water to obtain a maximum volume equals to 8µL. <br>
| |
| - |
| |
| - | Ligation mix (NEB): <br>
| |
| - | 3µL of T4 ligase + 3µL of T4 ligase buffer. <br>
| |
| - | Add 2µL / tube. <br>
| |
| - | Incubation 1h at RT. <br>
| |
| - |
| |
| - | ==== Electroporation ====
| |
| - |
| |
| - | Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br>
| |
| - |
| |
| - | Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br>
| |
"
