User:DavidC/8 October 2009

DNA extraction
Miniprep extraction (promega) of: BBa_B0030 + BBa_C0012 + BBa_B0014; BBa_P1001 + BBa_B0014; BBa_B0014 + BBa_P1003 + BBa_B0014;

Ligation between BBa_I712669 + BBa_B0014 and BBa_B0014 + BBa_P1003 + BBa_B0014
Restriction of BBa_I712669 + BBa_B0014 by EcoRI and SpeI (1475bp):

DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Spe I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_B0014 + BBa_P1003 + BBa_B0014 by EcoRI and XbaI (3770bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Xba I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_C0012 + BBa_B0014 by SpeI and PstI (3302bp):
DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Spe I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 by SpeI and EcoRI (1279bp):
DNA (miniprep) = 30µL Buffer H (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Spe I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Ligation between BBa_B0030 + BBa_C0012 + BBa_B0014 and BBa_P1001 + BBa_B0014:
Restriction digest of BBa_B0030 + BBa_C0012 + BBa_B0014 by EcoRI and SpeI (3217bp): DNA (miniprep) = 30µL Buffer EcoRI (NEB) = 4µL H20 = 4µL Eco RI (NEB) = 1µL Spe I (NEB) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 + BBa_B0014 by EcoRI and XbaI (1374bp):

DNA (miniprep) = 30µL Buffer M (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Xba I (TAKARA) = 1µL 1 hour of incubation at 37°C.

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

Samples: BBa_P1001, BBa_B0030 (EcoRI/XbaI), BBa_C0012 + BBa_B0014.



Samples : BBa_P1001 + BBa_B0014, BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_B0014 + BBa_P1003 + BBa_B0014, BBa_I712669 + BBa_B0014.



DNA purification
Kit Qiagen “gel extraction kit”, final volume = 50µL.

Cell culture
Growth of bacteria in 5mL of LB medium and 5µL of the correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL)

Samples: BBa_P1001 + BBaB0014; BBa_P1003 + BBa_B0014; BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_R0040; BBa_C0040; BBa_B0014; BBa_B0030; BBa_P1001.