User:DavidC/10 October 2009
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+ | === Saturday the 10th === | ||
+ | |||
+ | ==== DNA extraction === | ||
+ | |||
+ | Plasmid extraction by using minipreps (promega): <br> | ||
+ | BBa_B0014; <br> | ||
+ | BBa_E0240; <br> | ||
+ | BBa_B0030; <br> | ||
+ | BBa_R0010; <br> | ||
+ | p53. <br> | ||
+ | |||
+ | ==== Cell culture ==== | ||
+ | |||
+ | Growth of <i>E.coli</i> DH5alpha into 500mL of LB medium with 500µL of ampicillin = 50mg/mL. <br><br> | ||
+ | |||
+ | Samples: <br> | ||
+ | p53 (2 times); <br> | ||
+ | |||
+ | ==== Restriction ==== | ||
+ | |||
+ | ==== Ligation between BBa_R0010 with BBa_B0030: ==== | ||
+ | |||
+ | Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp): <br> | ||
+ | |||
+ | DNA (miniprep) = 30µL <br> | ||
+ | Buffer M (TAKARA) = 4µL <br> | ||
+ | H20 = 4µL <br> | ||
+ | EcoRI (TAKARA) = 1µL <br> | ||
+ | XbaI (TAKARA) = 1µL <br> | ||
+ | 1 hour of incubation at 37°C. <br><br> | ||
+ | |||
+ | Restriction digest of BBa_R0010 by EcoRI and SpeI (200bp): <br> | ||
+ | |||
+ | DNA (miniprep) = 30µL <br> | ||
+ | Buffer EcoRI (NEB) = 13µL <br> | ||
+ | H20 = 13µL <br> | ||
+ | EcoRI (NEB) = 1µL <br> | ||
+ | SpeI (NEB) = 1µL <br> | ||
+ | 1 hour of incubation at 37°C. <br> | ||
+ | |||
+ | ==== Electrophoresis ==== | ||
+ | |||
+ | ==== DNA electrophoresis ==== | ||
+ | |||
+ | 85 Volt, 15 minutes. <br> | ||
+ | 105 Volt, 40 minutes. <br> | ||
+ | Ladder fermentas 1 Kb. <br> | ||
+ | |||
+ | Samples: BBa_B0030, BBa_R0010. | ||
+ | <br> | ||
+ | |||
+ | [[image:SA1010.png|center]] | ||
+ | <br> | ||
+ | |||
+ | ==== Ligation ==== | ||
+ | |||
+ | ==== Ligation between BBa_R0010 and BB_B0030 ==== | ||
+ | |||
+ | First report: <br> | ||
+ | Plasmid (B0030) = 2µL <br> | ||
+ | Insert (R0010) = 8µL <br> | ||
+ | Solution A = 12µL <br> | ||
+ | Solution B = 3µL <br><br> | ||
+ | |||
+ | Second report: <br> | ||
+ | Plasmid (B0030) = 2µL <br> | ||
+ | Insert (R0010) = 6µL <br> | ||
+ | Solution A = 6µL <br> | ||
+ | Solution B = 2µL <br> | ||
+ | |||
+ | ==== Ligation between BBa_P1001 and BBa_B0014 ==== | ||
+ | |||
+ | First report: <br> | ||
+ | Plasmid (P1001) = 2µL <br> | ||
+ | Insert (B0014) = 10µL <br> | ||
+ | Solution A = 12µL <br> | ||
+ | Solution B = 4µL <br> | ||
+ | |||
+ | ==== Electroporation ==== | ||
+ | |||
+ | Electroporation cuvettes = 2mm ; inoculums of electrocompetent <i>E.coli</i> DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation. <br> | ||
+ | |||
+ | Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL). <br> | ||
+ | |||
+ | ==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030: ==== | ||
+ | |||
+ | First report: <br> | ||
+ | Plasmid (C0012 + B0014) = 2µL <br> | ||
+ | Insert (B0030) = 10µL <br> | ||
+ | Solution A = 12µL <br> | ||
+ | Solution B = 4µL <br> |
Latest revision as of 17:27, 21 October 2009
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