User:DavidC/9 September 2009

Ligation between BBa_P1002 and BBa_B0014:
BBa_B0014 (concentration = 3,70µg/µL) restriction digest by EcoRI and XbaI (3284pb):

DNA (10µg final) = 2,7µL Buffer Eco R1 (NEB) = 5µL H20 = 41,3µL 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.

BBa_P1002 (concentration = 2,86µg/µL) restriction digest by EcoRI and SpeI (943bp):

DNA (10µg final) = 3,5µL Buffer Eco R1 (NEB) = 5µL BSA = 0,5µL H20 = 39µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

Ligation between BBa_C0012 and BBa_B0014:
BBa_B0014 (concentration = 3,70µg/µL)restriction digest by EcoRI and XbaI (3284bp):

Same sample use for ligation between P1002 and B0014.

BBa_C0012 (concentration = 1,49µg/µL) restriction digest by EcoRI and SpeI (1128bp):

DNA (10µg final) = 6,7µL Buffer Eco R1 (NEB) = 5µL H20 = 35,8µL BSA = 0,5µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

Restriction digest of BBa_C0040 (concentration = 4,35µg/µL) by EcoRI and SpeI (660bp):
DNA (10µg final)= 2,3µL Buffer Eco R1 (NEB) = 5µL H20 = 40,7µL Eco R1 (NEB) = 1µL Spe 1 (NEB) = 1µL Incubation 1h at 37°C.

DNA electrophoresis
85 Volt, 15 minutes. 105 Volt, 40 minutes. Ladder fermentas 1 Kb. Samples: BBa_C0012, BBa_C0040, BBa_B0014, BBa_P1002.



Ligation
Ligation pairs, plasmid / insert (respectively):

BBa_B0014/ BBa_C0012 BBa_B0014/BBa_P1002

First ratio: Plasmid = 1µL Insert = 4µL

Second ratio: Plasmid = 1µL Insert = 2µL

Third ratio: Plasmid = 2µL Insert = 4µL

Fourth ratio: Plasmid = 1µL Insert = 5µL

Add sterilized water to maximum volume equal to 8µL.

Ligation mix : 9µL T4 ligase + 9µL T4 ligase buffer. Add 2µL / tube. Incubate 1h at RT.

Electroporation
Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.

Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).