User:DavidC/18 September 2009
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Friday the 18th
Restriction digest
Ligation between BBa_B0014 and BBa_P1003
Restriction digest of BBa_B0014 (= 2,41µg/µL) 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.
Restriction digest of BBa_P1003 (4,17µg/µL) by EcoRI and SpeI (967bp):
DNA (10µg final) = 2,4µL
Buffer Eco R1 (NEB) = 5µL
H20 = 40,6µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
Ligation beween BBa_B0014 and BBa_C0012
BBa_B0014 (= 2,41µg/µL) restriction digest by EcoRI and XbaI (3284bp):
Same samples as the restriction digest used for B0014 and P1003 ligation.
BBa_C0012 (1,56µg/µL) restriction digest by EcoRI and SpeI (1128bp):
DNA (10µg final) = 6,4µL
Buffer Eco R1 (NEB) = 5µL
H20 = 36,1µL
BSA = 0,5µL
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_P1003, BBa_B0014, BBa_C0012.
DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.
Ligation
Ligation schemes: plasmid / insert:
BBa_B0014/BBa_P1003 ;
BBa_B0014/BBa_C0012.
First report:
Plasmid = 1µL
Insert = 5µL
Second report:
Plasmid = 1µL
Insert = 3µL
Third report:
Plasmid = 1µL
Insert = 7µL
NEB Enzymes
For each samples add sterilized water to obtain a maximum volume equals to 8µL.
Ligation mix (NEB):
3µL of T4 ligase + 3µL of T4 ligase buffer.
Add 2µL / tube.
Incubation 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).