User:DavidC/18 September 2009

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).