User:DavidC/10 October 2009

DNA extraction
Plasmid extraction by using minipreps (promega): BBa_B0014; BBa_E0240; BBa_B0030; BBa_R0010; p53.

Cell culture
Growth of E.coli DH5alpha into 500mL of LB medium with 500µL of ampicillin = 50mg/mL.

Samples: p53 (2 times);

Ligation between BBa_R0010 with BBa_B0030:
Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) = 4µL H20 = 4µL EcoRI (TAKARA) = 1µL XbaI (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_R0010 by EcoRI and SpeI (200bp):

DNA (miniprep) = 30µL Buffer EcoRI (NEB) = 13µL H20 = 13µL EcoRI (NEB) = 1µL SpeI (NEB) = 1µL 1 hour of incubation at 37°C.

DNA electrophoresis
85 Volt, 15 minutes. 105 Volt, 40 minutes. Ladder fermentas 1 Kb.

Samples: BBa_B0030, BBa_R0010.



Ligation between BBa_R0010 and BB_B0030
First report: Plasmid (B0030) = 2µL Insert (R0010) = 8µL Solution A = 12µL Solution B = 3µL

Second report: Plasmid (B0030) = 2µL Insert (R0010) = 6µL Solution A = 6µL Solution B = 2µL

Ligation between BBa_P1001 and BBa_B0014
First report: Plasmid (P1001) = 2µL Insert (B0014) = 10µL Solution A = 12µL Solution B = 4µL

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).

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030:
First report: Plasmid (C0012 + B0014) = 2µL Insert (B0030) = 10µL Solution A = 12µL Solution B = 4µL