User:DavidC/9 September 2009
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Wednesday the 9th
Restriction digest:
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).