User:DavidC/8 October 2009

From 2009.igem.org

(Difference between revisions)
(DNA electrophoresis)
(DNA electrophoresis)
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Samples: BBa_P1001, BBa_B0014, BBa_C0012 + BBa_B0014.
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Samples: BBa_P1001, BBa_B0030 (EcoRI/XbaI), BBa_C0012 + BBa_B0014.
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Samples : BBa_P1001 + BBa_B0014, BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_I712669 + BBa_B0014.
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Samples : BBa_P1001 + BBa_B0014, BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_B0014 + BBa_P1003 + BBaB0014, BBa_I712669 + BBa_B0014.
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Revision as of 17:20, 21 October 2009

framless



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Contents

Thursday the 8th

DNA extraction

Miniprep extraction (promega) of:
BBa_B0030 + BBa_C0012 + BBa_B0014;
BBa_P1001 + BBa_B0014;
BBa_B0014 + BBa_P1003 + BBa_B0014;

Restriction digest

Ligation between BBa_I712669 + BBa_B0014 and BBa_B0014 + BBa_P1003 + BBa_B0014

Restriction of BBa_I712669 + BBa_B0014 by EcoRI and SpeI (1475bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_B0014 + BBa_P1003 + BBa_B0014 by EcoRI and XbaI (3770bp):

DNA (miniprep) = 30µL
Buffer M (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Xba I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_C0012 + BBa_B0014 by SpeI and PstI (3302bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 by SpeI and EcoRI (1279bp):

DNA (miniprep) = 30µL
Buffer H (TAKARA) = 4µL
H20 = 4µL
Eco RI (TAKARA) = 1µL
Spe I (TAKARA) = 1µL
1 hour of incubation at 37°C.

Ligation between BBa_B0030 + BBa_C0012 + BBa_B0014 and BBa_P1001 + BBa_B0014:

Restriction digest of BBa_B0030 + BBa_C0012 + BBa_B0014 by EcoRI and SpeI (3217bp):
DNA (miniprep) = 30µL
Buffer EcoRI (NEB) = 4µL
H20 = 4µL
Eco RI (NEB) = 1µL
Spe I (NEB) = 1µL
1 hour of incubation at 37°C.

Restriction digest of BBa_P1001 + BBa_B0014 by EcoRI and XbaI (1374bp):

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_B0030 (EcoRI/XbaI), BBa_C0012 + BBa_B0014.

TH0810(1).png


Samples : BBa_P1001 + BBa_B0014, BBa_B0030 + BBa_C0012 + BBa_B0014, BBa_B0014 + BBa_P1003 + BBaB0014, BBa_I712669 + BBa_B0014.

TH0810(2).png


DNA purification

Kit Qiagen “gel extraction kit”, final volume = 50µL.

Cell culture

Growth of bacteria in 5mL of LB medium and 5µL of the correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL)

Samples:
BBa_P1001 + BBaB0014;
BBa_P1003 + BBa_B0014;
BBa_B0030 + BBa_C0012 + BBa_B0014,
BBa_R0040;
BBa_C0040;
BBa_B0014;
BBa_B0030;
BBa_P1001.