User:DavidC/10 October 2009
From 2009.igem.org
Saturday the 10th
= 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);
Restriction
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.
Electrophoresis
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples: BBa_B0030, BBa_R0010.
Ligation
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