User:DavidC/6 October 2009

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=== Tuesday the 6th ===
 +
 +
==== DNA extraction ====
 +
 +
Miniprep extraction (promega) of: <br>
 +
 +
BBa_B0014; <br>
 +
BBa_B0030; <br>
 +
BBa_P1001; <br>
 +
BBa_C0040; <br>
 +
BBa_R0040. <br>
 +
 +
==== Restriction digest ====
 +
 +
==== Ligation between BBa_B0014 and BBa_P1001: ====
 +
 +
Restriction digest of BBa_B0014 by PstI and SpeI (3284pb): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer (NEB) = 5µL <br>
 +
H20 = 13µL <br>
 +
PstI (NEB) = 1µL <br>
 +
Spe I (NEB) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_P1001 by PstI and XbaI (1279bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 ====
 +
 +
Restriction digest of BBa_C0012 + BBa_B0014 by EcoRI and SpeI (1223bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer (NEB) = 5µL <br>
 +
H20 = 13µL <br>
 +
PstI (NEB) = 1µL <br>
 +
Spe I (NEB) = 1µL <br>
 +
1 hour of incubation at 37°C. <br><br>
 +
 +
Restriction digest of BBa_B0030 by EcoRI and XbaI (2094bp): <br>
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Restriction digest of BBa_C0040 by Eco RI and Spe I (660bp) ====
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Spe I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== Restriction of BBa_R0040 by Eco RI and Xba I (2133bp) ====
 +
 +
DNA (miniprep) = 30µL <br>
 +
Buffer M (TAKARA) = 4µL <br>
 +
H20 = 4µL <br>
 +
Eco RI (TAKARA) = 1µL <br>
 +
Xba I (TAKARA) = 1µL <br>
 +
1 hour of incubation at 37°C. <br>
 +
 +
==== DNA electrophoresis ====
 +
 +
85 Volt, 15 minutes. <br>
 +
105 Volt, 40 minutes. <br>
 +
Ladder fermentas 1 Kb. <br>
 +
 +
 +
Samples:
 +
 +
==== DNA purification ====
 +
 +
Kit Qiagen “gel extraction kit”, final volume = 50µL. <br>
 +
 +
==== Ligation ====
 +
 +
Ligation of BBa_B0014 with BBa_P1001 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report: <br>
 +
Plasmid (P1001) = 1µL <br>
 +
Insert (B0014) = 7µL <br>
 +
Solution A = 1µL <br>
 +
Solution B = 1µL <br>
 +
 +
Second report: <br>
 +
Plasmid (P1001) = 0,5µL <br>
 +
Insert (B0014) = 4µL <br>
 +
Solution A = 4,5µL <br>
 +
Solution B = 1µL <br>
 +
 +
Ligation between BBa_C0012 + BBa_B0014 and BBa_B0030 (TAKARA, DNA ligation kit) : <br>
 +
 +
First report: <br>
 +
Plasmid (B0030) = 1µL <br>
 +
Insert (C0012 + B0014) = 7µL <br>
 +
Solution A = 1µL <br>
 +
Solution B = 1µL <br>
 +
 +
Second report: <br>
 +
Plasmid (B0030) = 0,5µL <br>
 +
Insert (C0012 + B0014) = 4µL <br>
 +
Solution A = 4,5µL <br>
 +
Solution B = 1µL <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>

Revision as of 04:14, 21 October 2009

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Contents

Tuesday the 6th

DNA extraction

Miniprep extraction (promega) of:

BBa_B0014;
BBa_B0030;
BBa_P1001;
BBa_C0040;
BBa_R0040.

Restriction digest

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:

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).

Retrieved from "http://2009.igem.org/User:DavidC/6_October_2009"