From 2009.igem.org
| August
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| M | T | W | T | F | S | S
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| [http://2009.igem.org/User:DavidC/1_August_2009 1]
| [http://2009.igem.org/User:DavidC/2_August_2009 2]
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| [http://2009.igem.org/User:DavidC/3_August_2009 3]
| [http://2009.igem.org/User:DavidC/4_August_2009 4]
| [http://2009.igem.org/User:DavidC/5_August_2009 5]
| [http://2009.igem.org/User:DavidC/6_August_2009 6]
| [http://2009.igem.org/User:DavidC/7_August_2009 7]
| [http://2009.igem.org/User:DavidC/8_August_2009 8]
| [http://2009.igem.org/User:DavidC/9_August_2009 9]
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| [http://2009.igem.org/User:DavidC/10_August_2009 10]
| [http://2009.igem.org/User:DavidC/11_August_2009 11]
| [http://2009.igem.org/User:DavidC/12_August_2009 12]
| [http://2009.igem.org/User:DavidC/13_August_2009 13]
| [http://2009.igem.org/User:DavidC/14_August_2009 14]
| [http://2009.igem.org/User:DavidC/15_August_2009 15]
| [http://2009.igem.org/User:DavidC/16_August_2009 16]
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| [http://2009.igem.org/User:DavidC/17_August_2009 17]
| [http://2009.igem.org/User:DavidC/18_August_2009 18]
| [http://2009.igem.org/User:DavidC/19_August_2009 19]
| [http://2009.igem.org/User:DavidC/20_August_2009 20]
| [http://2009.igem.org/User:DavidC/21_August_2009 21]
| [http://2009.igem.org/User:DavidC/22_August_2009 22]
| [http://2009.igem.org/User:DavidC/23_August_2009 23]
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| [http://2009.igem.org/User:DavidC/24_August_2009 24]
| [http://2009.igem.org/User:DavidC/25_August_2009 25]
| [http://2009.igem.org/User:DavidC/26_August_2009 26]
| [http://2009.igem.org/User:DavidC/27_August_2009 27]
| [http://2009.igem.org/User:DavidC/28_August_2009 28]
| [http://2009.igem.org/User:DavidC/29_August_2009 29]
| [http://2009.igem.org/User:DavidC/30_August_2009 30]
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| [http://2009.igem.org/User:DavidC/31_August_2009 31]
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| September
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| M | T | W | T | F | S | S
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| [http://2009.igem.org/User:DavidC/1_September_2009 1]
| [http://2009.igem.org/User:DavidC/2_September_2009 2]
| [http://2009.igem.org/User:DavidC/3_September_2009 3]
| [http://2009.igem.org/User:DavidC/4_September_2009 4]
| [http://2009.igem.org/User:DavidC/5_September_2009 5]
| [http://2009.igem.org/User:DavidC/6_September_2009 6]
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| [http://2009.igem.org/User:DavidC/7_September_2009 7]
| [http://2009.igem.org/User:DavidC/8_September_2009 8]
| [http://2009.igem.org/User:DavidC/9_September_2009 9]
| [http://2009.igem.org/User:DavidC/10_September_2009 10]
| [http://2009.igem.org/User:DavidC/11_September_2009 11]
| [http://2009.igem.org/User:DavidC/12_September_2009 12]
| [http://2009.igem.org/User:DavidC/13_September_2009 13]
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| [http://2009.igem.org/User:DavidC/14_September_2009 14]
| [http://2009.igem.org/User:DavidC/15_September_2009 15]
| [http://2009.igem.org/User:DavidC/16_September_2009 16]
| [http://2009.igem.org/User:DavidC/17_September_2009 17]
| [http://2009.igem.org/User:DavidC/18_September_2009 18]
| [http://2009.igem.org/User:DavidC/19_September_2009 19]
| [http://2009.igem.org/User:DavidC/20_September_2009 20]
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| [http://2009.igem.org/User:DavidC/21_September_2009 21]
| [http://2009.igem.org/User:DavidC/22_September_2009 22]
| [http://2009.igem.org/User:DavidC/23_September_2009 23]
| [http://2009.igem.org/User:DavidC/24_September_2009 24]
| [http://2009.igem.org/User:DavidC/25_September_2009 25]
| [http://2009.igem.org/User:DavidC/26_September_2009 26]
| [http://2009.igem.org/User:DavidC/27_September_2009 27]
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| [http://2009.igem.org/User:DavidC/28_September_2009 28]
| [http://2009.igem.org/User:DavidC/29_September_2009 29]
| [http://2009.igem.org/User:DavidC/30_September_2009 30]
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| October
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| M | T | W | T | F | S | S
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| [http://2009.igem.org/User:DavidC/1_October_2009 1]
| [http://2009.igem.org/User:DavidC/2_October_2009 2]
| [http://2009.igem.org/User:DavidC/3_October_2009 3]
| [http://2009.igem.org/User:DavidC/4_October_2009 4]
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| [http://2009.igem.org/User:DavidC/5_October_2009 5]
| [http://2009.igem.org/User:DavidC/6_October_2009 6]
| [http://2009.igem.org/User:DavidC/7_October_2009 7]
| [http://2009.igem.org/User:DavidC/8_October_2009 8]
| [http://2009.igem.org/User:DavidC/9_October_2009 9]
| [http://2009.igem.org/User:DavidC/10_October_2009 10]
| [http://2009.igem.org/User:DavidC/11_October_2009 11]
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| [http://2009.igem.org/User:DavidC/12_October_2009 12]
| [http://2009.igem.org/User:DavidC/13_October_2009 13]
| [http://2009.igem.org/User:DavidC/14_October_2009 14]
| [http://2009.igem.org/User:DavidC/15_October_2009 15]
| [http://2009.igem.org/User:DavidC/16_October_2009 16]
| [http://2009.igem.org/User:DavidC/17_October_2009 17]
| [http://2009.igem.org/User:DavidC/18_October_2009 18]
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| [http://2009.igem.org/User:DavidC/19_October_2009 19]
| [http://2009.igem.org/User:DavidC/20_October_2009 20]
| [http://2009.igem.org/User:DavidC/21_October_2009 21]
| [http://2009.igem.org/User:DavidC/22_October_2009 22]
| [http://2009.igem.org/User:DavidC/23_October_2009 23]
| [http://2009.igem.org/User:DavidC/24_October_2009 24]
| [http://2009.igem.org/User:DavidC/25_October_2009 25]
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| [http://2009.igem.org/User:DavidC/26_October_2009 26]
| [http://2009.igem.org/User:DavidC/27_October_2009 27]
| [http://2009.igem.org/User:DavidC/28_October_2009 28]
| [http://2009.igem.org/User:DavidC/29_October_2009 29]
| [http://2009.igem.org/User:DavidC/30_October_2009 30]
| [http://2009.igem.org/User:DavidC/31_October_2009 31]
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Wednesday the 9th
Restriction digest:
Ligation between BBa_P1002 and BBa_B0014:
BBa_B0014 (concentration = 3,70µg/µL) restriction digest by EcoRI and XbaI (3284pb):
DNA (10µg final) = 2,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 41,3µL
Eco R1 = 1µL
Incubation 1h at 37°C.
DNA purification with a nucleospin (macherey-nagel).
DNA = 50µL
Buffer 2 (NEB) = 6µL
BSA (NEB) = 0,6µL
H20 = 2,4µL
Xba 1 = 1µL
Incubation 1h at 37°C.
BBa_P1002 (concentration = 2,86µg/µL) restriction digest by EcoRI and SpeI (943bp):
DNA (10µg final) = 3,5µL
Buffer Eco R1 (NEB) = 5µL
BSA = 0,5µL
H20 = 39µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
Ligation between BBa_C0012 and BBa_B0014:
BBa_B0014 (concentration = 3,70µg/µL)restriction digest by EcoRI and XbaI (3284bp):
Same sample use for ligation between P1002 and B0014.
BBa_C0012 (concentration = 1,49µg/µL) restriction digest by EcoRI and SpeI (1128bp):
DNA (10µg final) = 6,7µL
Buffer Eco R1 (NEB) = 5µL
H20 = 35,8µL
BSA = 0,5µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
Restriction digest of BBa_C0040 (concentration = 4,35µg/µL) by EcoRI and SpeI (660bp):
DNA (10µg final)= 2,3µL
Buffer Eco R1 (NEB) = 5µL
H20 = 40,7µL
Eco R1 (NEB) = 1µL
Spe 1 (NEB) = 1µL
Incubation 1h at 37°C.
DNA electrophoresis
85 Volt, 15 minutes.
105 Volt, 40 minutes.
Ladder fermentas 1 Kb.
Samples: BBa_C0012, BBa_C0040, BBa_B0014, BBa_P1002.
Ligation
Ligation pairs, plasmid / insert (respectively):
BBa_B0014/ BBa_C0012
BBa_B0014/BBa_P1002
First ratio:
Plasmid = 1µL
Insert = 4µL
Second ratio:
Plasmid = 1µL
Insert = 2µL
Third ratio:
Plasmid = 2µL
Insert = 4µL
Fourth ratio:
Plasmid = 1µL
Insert = 5µL
Add sterilized water to maximum volume equal to 8µL.
Ligation mix :
9µL T4 ligase + 9µL T4 ligase buffer.
Add 2µL / tube.
Incubate 1h at RT.
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).
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