User:DavidC/16 October 2009

Tuesday the 13th to Sunday the 18th
Growth of bacteria in the goal to extract the bio-bricks:

BBa_J23100 + BBa_B0014; BBa_B0030 + BBa_J23100 + BBa_B0014; BBa_R0010 + BBa_B0030; BBa_P1003 + BBa_B0014; BBa_B0014 + BBa_P1003 + BBa_B0014; BBa_C0012 + BBa_B0014; BBa_B0030 + BBa_C0012 + BBa_B0014; BBa_I712669 + BBa_B0014;

5mL of LB medium + 5µL of correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL).

Ligation between D protein and pSB1A2
By using the In-Fusion team from TAKARA.

Plasmid pSB1A2 = 5µL

Insert, D protein (PCR product)= 6µL 5X In-Fusion Reaction Buffer = 3µL In-Fusion Enzyme = 1µL dH2O = 0µL TOTAL = 10µL

Incubation in a thermocycler : 15 minutes at 37°C, followed by 15 minutes at 50°C.

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).

After 72 hours of incubation we obtain a colony. Atfer inoculation in 5mL of LB with 5µL of ampicillin we extract the plasmid with a miniprep.