From 2009.igem.org
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| - | Samples: | + | Samples: BBa_P1001, BBa_B0014, BBa_C0040, BBa_R0040, BBa_B0030, BBa_C0012 + BBa_B0014. |
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| | + | [[image:TU0610.png|center]] |
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| | ==== DNA purification ==== | | ==== DNA purification ==== |
Latest revision as of 16:13, 21 October 2009
| August
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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]
|
| [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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Tuesday the 6th
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.
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).
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