User:DavidC/6 October 2009

DNA extraction
Miniprep extraction (promega) of:

BBa_B0014; BBa_B0030; BBa_P1001; BBa_C0040; BBa_R0040.

Ligation between BBa_B0014 and BBa_P1001:
Restriction digest of BBa_B0014 by PstI and SpeI (3284pb):

DNA (miniprep) = 30µL Buffer (NEB) = 5µL H20 = 13µL PstI (NEB) = 1µL Spe I (NEB) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 by PstI and XbaI (1279bp):

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

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030
Restriction digest of BBa_C0012 + BBa_B0014 by EcoRI and SpeI (1223bp):

DNA (miniprep) = 30µL Buffer (NEB) = 5µL H20 = 13µL PstI (NEB) = 1µL Spe I (NEB) = 1µL 1 hour of incubation at 37°C.

Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp):

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

Restriction digest of BBa_C0040 by Eco RI and Spe I (660bp)
DNA (miniprep) = 30µL Buffer M (TAKARA) = 4µL H20 = 4µL Eco RI (TAKARA) = 1µL Spe I (TAKARA) = 1µL 1 hour of incubation at 37°C.

Restriction of BBa_R0040 by Eco RI and Xba I (2133bp)
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_B0014, BBa_C0040, BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.



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

Ligation
Ligation of BBa_B0014 with BBa_P1001 (TAKARA, DNA ligation kit) :

First report: Plasmid (P1001) = 1µL Insert (B0014) = 7µL Solution A = 1µL Solution B = 1µL

Second report: Plasmid (P1001) = 0,5µL Insert (B0014) = 4µL Solution A = 4,5µL Solution B = 1µL

Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 (TAKARA, DNA ligation kit) :

First report: Plasmid (B0030) = 1µL Insert (C0012 + B0014) = 7µL Solution A = 1µL Solution B = 1µL

Second report: Plasmid (B0030) = 0,5µL Insert (C0012 + B0014) = 4µL Solution A = 4,5µL Solution B = 1µL

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).