Team:British Columbia/Notebook

From 2010.igem.org

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<h3>Standard Operating Protocols (SOPS)</h3>
<h3>Standard Operating Protocols (SOPS)</h3>
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<h4>Colony PCR</h4>
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Supplies Needed:
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<ol><li>PCR tubes</li><li>BioBrick PCR primers (G1004, G1005) or (VF2, VR)</li><li>Taq Polymerase</li><li>10x Reaction Buffer</li><li>10mM dNTPs</li><li>sdH2O</li><li>Colonies to be PCR’ed</li><li>Agar plate for indexing</li></ol>
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Steps:
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<ol><li>Make master mix of primers and other PCR components EXCEPT Taq polymerase. Keep on ice.</li>
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<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption> PCR Master mix</caption>
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<tr><td>Reagent<td colspan="2">1x rxn volume (uL)<td>Master Mix</td></tr>
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<tr><td>5x rxn buffer<td>5<td>xn<td></td></tr>
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<tr><td>10mM dNTP<td>0.5<td>xn<td></td></tr>
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<tr><td>sdH2O<td>9.15<td>xn<td></td></tr>
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<tr><td>Phusion polymerase<td>0.1<td>xn<td></td></tr>
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<tr><td>MgCl2<td>2<td>xn<td></td></tr>
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<tr><td>DMSO - 5%<td>1.25<td>xn<td></td></tr>
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<tr><td>gDNA<td>3<td>xn<td></td></tr>
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<tr><td>Total<td>25</td></tr></table></div>
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N = number of PCR tubes/samples
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Make sure to add about 2 extra samples to account for pipetting error and 1 extra sample for water control.
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Example: for 20 colonies, let N be: 20 (colonies) + 2 (extra)+ 1 (water) = 23
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Revision as of 21:54, 27 October 2010



Notebook: Need to Know

Welcome to our wiki notebook! We have organized our notebook according to sub-teams. Each page will provide you with a link to our actual notebook on OpenWetWare. To deliver the essentials here on the wiki (so you don't have to read through 6 months of experiments to get our message), we discuss the protocols, experimental outline, troubleshooting and optimization, and potential implications for iGEM.





Standard Operating Protocols (SOPS)

Colony PCR

Supplies Needed:
  1. PCR tubes
  2. BioBrick PCR primers (G1004, G1005) or (VF2, VR)
  3. Taq Polymerase
  4. 10x Reaction Buffer
  5. 10mM dNTPs
  6. sdH2O
  7. Colonies to be PCR’ed
  8. Agar plate for indexing
Steps:
  1. Make master mix of primers and other PCR components EXCEPT Taq polymerase. Keep on ice.
  2. PCR Master mix
    Reagent1x rxn volume (uL)Master Mix
    5x rxn buffer5xn
    10mM dNTP0.5xn
    sdH2O9.15xn
    Phusion polymerase0.1xn
    MgCl22xn
    DMSO - 5%1.25xn
    gDNA3xn
    Total25
    N = number of PCR tubes/samples Make sure to add about 2 extra samples to account for pipetting error and 1 extra sample for water control. Example: for 20 colonies, let N be: 20 (colonies) + 2 (extra)+ 1 (water) = 23


OpenWetWare (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.


See our UBC OWW notebook.