From 2009.igem.org
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| 5mL of LB medium + 5µL of correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL). <br> | | 5mL of LB medium + 5µL of correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL). <br> |
| + | |
| + | |
| + | ==== Ligation between D protein and pSB1A2 ==== |
| + | |
| + | By using the In-Fusion team from TAKARA. <br> |
| + | |
| + | Plasmid pSB1A2 = 5µL<br> |
| + | |
| + | Insert, D protein (PCR product)= 6µL <br> |
| + | 5X In-Fusion Reaction Buffer = 3µL <br> |
| + | In-Fusion Enzyme = 1µL <br> |
| + | dH2O = 0µL <br> |
| + | TOTAL = 10µL <br> |
| + | |
| + | Incubation in a thermocycler : <br> |
| + | 15 minutes at 37°C, followed by 15 minutes at 50°C. <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> |
| + | |
| + | After 72 hours of incubation we obtain a colony. Atfer inoculation in 5mL of LB with 5µL of ampicillin we extract the plasmid with a miniprep. |
Latest revision as of 04:38, 21 October 2009
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]
|
[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]
|
[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]
|
[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]
|
[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 13th to Sunday the 18th
Growth of bacteria in the goal to extract the bio-bricks:
BBa_J23100 + BBa_B0014;
BBa_B0030 + BBa_J23100 + BBa_B0014;
BBa_R0010 + BBa_B0030;
BBa_P1003 + BBa_B0014;
BBa_B0014 + BBa_P1003 + BBa_B0014;
BBa_C0012 + BBa_B0014;
BBa_B0030 + BBa_C0012 + BBa_B0014;
BBa_I712669 + BBa_B0014;
5mL of LB medium + 5µL of correct antibiotic (kanamycin = 50mg/mL, ampicillin = 50mg/mL and tetracycline = 12,5mg/mL).
Ligation between D protein and pSB1A2
By using the In-Fusion team from TAKARA.
Plasmid pSB1A2 = 5µL
Insert, D protein (PCR product)= 6µL
5X In-Fusion Reaction Buffer = 3µL
In-Fusion Enzyme = 1µL
dH2O = 0µL
TOTAL = 10µL
Incubation in a thermocycler :
15 minutes at 37°C, followed by 15 minutes at 50°C.
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).
After 72 hours of incubation we obtain a colony. Atfer inoculation in 5mL of LB with 5µL of ampicillin we extract the plasmid with a miniprep.