Team:Paris Bettencourt/Notebook/TB-ception/Tuesday 10th September.html

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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/TB-ception" target="_blank" class="tbnotelogo TClogo"> ASDF </a>
<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/TB-ception" target="_blank" class="tbnotelogo TClogo"> ASDF </a>
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<h3>Tuesday 10<sup>th</sup> July</h3>
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<h3>Tuesday 10<sup>th</sup> September</h3>
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PCR for the pSB3C5 and pSB1C3 backbone with the oligos for building a new backbone. The oligos are --> pDCA reverse with pDCA1/2/3 forward; pDCo4 reverse and forward. Gel slice and gel purification.
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<br />• PCR for the linearized backbone: template - pSB1C3 (1 µL), primers (SB-prep-2Eb, SB-prep-3P-1; 2.5 µL), HE buffer (10 µL), dNTPs (1 µL), polymerase (0.5 µL), dH2O (32.5 µL).
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Passaged the macrophages.
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<br />
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program: 94.0 (30 s) - 94.0 (10 s), 55.0 (1 min), 72.0 (2 min), 72.0 (3 min) - 30 cycles.
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<br />• touchdown PCR for the new backbones:
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<table border="1">
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<tr>
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<td>Template</td>
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<td>Primers</td>
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</tr>
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<tr>
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<td>pACYC</td>
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<td>pDCA2 and pDCA</td>
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<td></td>
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</tr>
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<td>pET</td>
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<td>pDCo4-F and pDCo4-R</td>
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</tr>
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<td>pCDF</td>
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<td>pDCA1 and pDCA</td>
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</tr>
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<td>pColA</td>
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<td>pDCA3 and pDCA</td>
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</tr>
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</tr>
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</table>
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program: 95.0 (10 s) - 95.0 (30 s), 60.0 (30 s), 59.0 (30 s),58.0 (30 s), 56.0 (30 s), 55.0 (30 s), 72.0 (30 s) - 72.0 (10 min)- 30 cycles.
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Latest revision as of 12:53, 25 September 2013

TB-ception

Tuesday 10th September



• PCR for the linearized backbone: template - pSB1C3 (1 µL), primers (SB-prep-2Eb, SB-prep-3P-1; 2.5 µL), HE buffer (10 µL), dNTPs (1 µL), polymerase (0.5 µL), dH2O (32.5 µL).

program: 94.0 (30 s) - 94.0 (10 s), 55.0 (1 min), 72.0 (2 min), 72.0 (3 min) - 30 cycles.
• touchdown PCR for the new backbones:

Template Primers
pACYC pDCA2 and pDCA
pET pDCo4-F and pDCo4-R
pCDF pDCA1 and pDCA
pColA pDCA3 and pDCA


program: 95.0 (10 s) - 95.0 (30 s), 60.0 (30 s), 59.0 (30 s),58.0 (30 s), 56.0 (30 s), 55.0 (30 s), 72.0 (30 s) - 72.0 (10 min)- 30 cycles.

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