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User:DavidC/16 October 2009
From 2009.igem.org
Introduction Current therapies Gene therapies DVS Concept Tissue Vector Cell Vector Therapeutic plasmid Conclusion Implementation Tissue targeting Antitumor action Treatment modeling Conclusion
BioBricks Material & Method Bibliography
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.
