User:DavidC/8 October 2009
From 2009.igem.org
Thursday the 8th
DNA extraction
Miniprep extraction (promega) of:
BBa_B0030 + BBa_C0012 + BBa_B0014;
BBa_P1001 + BBa_B0014;
BBa_B0014 + BBa_P1003 + BBa_B0014;
Restriction digest
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.