User:DavidC/6 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: BBa_P1001, BBa_B0014, BBa_C0040, BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014.

TU0610.png


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).