User:DavidC/10 October 2009

From 2009.igem.org

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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>
 +
 +
==== 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>

Revision as of 04:27, 21 October 2009

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Contents

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.

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

Retrieved from "http://2009.igem.org/User:DavidC/10_October_2009"