Team:Warsaw/Calendar-Main/13 August 2009
From 2009.igem.org
(Difference between revisions)
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3.0 μl - primer BaxF (0.5 μM) | 3.0 μl - primer BaxF (0.5 μM) | ||
3.0 μl - primer BaxR2 (0.5 μM) | 3.0 μl - primer BaxR2 (0.5 μM) | ||
- | 1.0 μl - | + | 1.0 μl - template DNA |
32.0μl - mQ water | 32.0μl - mQ water | ||
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) | 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) | ||
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<li>Template DNA in 3 dilutions - 10<sup>1</sup> (pure plasmid DNA), 10<sup>-1</sup> and 10<sup>-2</sup>.</li> | <li>Template DNA in 3 dilutions - 10<sup>1</sup> (pure plasmid DNA), 10<sup>-1</sup> and 10<sup>-2</sup>.</li> | ||
<li>buffer, dNTP mix and polymerase from EURx.</li> | <li>buffer, dNTP mix and polymerase from EURx.</li> | ||
+ | <li>DNA template - human Bax with changed codons to be more efficient in yeast. </li> | ||
<br> | <br> | ||
<br> | <br> |
Revision as of 11:11, 14 August 2009
Amplyfing of Bax sequence
Justyna
Methods
- PCR reaction mix:
per 50μl:
5.0 μl - 10 x buffer (20mM MgSO4) 3.5 μl - dNTP mix (5mM) 3.0 μl - primer BaxF (0.5 μM) 3.0 μl - primer BaxR2 (0.5 μM) 1.0 μl - template DNA 32.0μl - mQ water 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
Thermal cycling conditions for PCR (~7-8 kb):
94°C, 1 min 0s 94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 0s cycles 1-10 94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 20s cycles 11-25 68°C, 7 min 0s 4°C, indefinite
Results:
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