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Team:Aix-Marseille/Notebook stab-construct
From 2014.igem.org
Notebook
Week 10 : 09/01/2014 ➡ 08/07/2014
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Construction of stability epitopes : the main idea is to use the primers 57-58 and 59 as matrices in order to synthesize and amplify the stability epitope. The PCR uses the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 55°C, the elongation time is 5", the number of cycle is 25.
Mix:
- 5µL OiGEM-57 or 58 or 59
- 2,5µL OiGEM-60
- 2,5µL OiGEM-44
- 25µL Q5 2x Mix
- 15µL H2O
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Clean-up of these PCR and elution with 30µL H2O at 70°C.
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Digestion ES of PCR products of the stability epitopes and of piGEM-01.13 (pSB1C3-BBa_J04450).
Mix:
- 10µL K10512-06 or 07 or 08 or 5µL piGEM-01.13
- 1µL EcoRI
- 1µL SpeI
- 5µL Buffer 2.1
- qsp 50µL of water
Incubation 60' at 37°C.
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Then, for K10512-06 or 07 or 08, inactivation of enzymes 20' at 80°C.
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Clean-up of piGEM-01.13 and elution with 30µL of water. Add 1µL of SAP (Promega) + 2µL of SAP buffer (dephosphorylation). Incubation 30' at 37°C, then Inactivation of enzyme 20' at 65°C.
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Ligation K10512-06 or 07 or 08 with piGEM-01.13.
Mix:
- 2µL piGEM-01.13
- 4µL K10512-06 or 07 or 08
- 1µL T4 ligase 10U/µL
- 2µL T4 ligase buffer
- 11µL H2O
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Transformation of 90µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.
Week 11 : 09/08/2014 ➡ 08/14/2014
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Test 5 clones of each transformation (with pSB1C3- K10512-06 or 07 or 08) by PCR. The temperature of annealing is 55°C, the elongation time is 1'
Mix:
- 2µL OiGEM-60
- 2µL OiGEM-44
- 4µL dNTP
- 1µL DMSO
- 2µL buffer
- 0,5µL Taq polymérase
- 8,5µL H2O
All clones are good except the K10512-08-2. The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.
