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| - | {{:Team:UC-SantaCruz_Template}}
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| - |
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| - | <p>
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| - | <strong>2013 UCSC IGEM TEAM Polar Tags Notebook.</strong>
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| - | </p>
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| - | <p>
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| - | <strong> </strong>
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| - | </p>
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| - | <p align="center">
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| - | <strong>Index-</strong>
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| - | </p>
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| - | <p>
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| - | <strong> </strong>
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| - | </p>
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| - | <p>
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| - | <strong><u>Topic Page #</u></strong>
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| - | </p>
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| - | <p>
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| - | <strong>Groups Goals </strong>
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| - | </p>
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| - | <p>
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| - | <strong>General PCR Protocol </strong>
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| - | </p>
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| - | <p>
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| - | <strong>General Agarose Gel Protocol-</strong>
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| - | </p>
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| - | <p>
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| - | <strong>General Chemicompetent Cell Transformation Protocol </strong>
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| - | </p>
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| - | <p>
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| - | <strong>LB Media Recipe</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Colony Pick Protocol</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Electroporation Protocol</strong>
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| - | </p>
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| - | <p>
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| - | <strong>DNA MW marker reference</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Reference Sequences </strong>
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| - | </p>
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| - | <p>
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| - | <strong>References </strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/6/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/7/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/8/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/9/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/13/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/15/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/16/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/19/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/20/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/21/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/22/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/25/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/26/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/27/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/28/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/29/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/30/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 8/31/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/1/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/2/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/3/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/4-5/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/6/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/7/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/8/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/10/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/11/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/12/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/13/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/14/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/15/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/16/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/17/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/18/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/19/13</strong>
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| - | </p>
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| - | <p>
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| - | <strong>Lab Notebook 9/20/13</strong>
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| - | </p>
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| - | <strong>
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| - | <br clear="all"/>
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| - | </strong>
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| - | <p>
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| - | <strong> </strong>
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| - | </p>
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| - | <p>
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| - | <strong> </strong>
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| - | </p>
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| - | <p align="center">
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| - | <strong>Tag Notebook</strong>
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| - | <img
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| - | width="207"
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| - | height="500"
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| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image001.jpg"
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| - | alt="https://lh6.googleusercontent.com/U_TIsAf8cmqlUEstyxEDc7HqNa14OLOjeNZXveeP6wfb5sCQOXIN9XIreASa_oFFpaZ4vqYghafm-Lq-rOCem4LJ2SBsUyfx42OG5goX09J6m1XjG0BWFOcp"
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| - | />
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| - | </p>
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| - | <p>
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| - | <strong>8/6/13</strong>
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| - | </p>
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| - | <p>
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| - | Purpose: To isolate and amplify PflI and Stpx polar markers from Caulobacter Crescentus (CB15N). Expected sizes are 614bp for PflI and 1.9kb for Stpx.
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| - | </p>
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| - | <p>
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| - | Each PCR sample contains:
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| - | </p>
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| - | <p>
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| - | - 23 ul MilliQ water
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| - | </p>
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| - | <p>
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| - | - 1 ul of template (colony pick from CB15 culture in PYE)
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| - | </p>
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| - | <p>
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| - | - 0.5 ul of 100 uM forward primer Stpx/PflI
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| - | </p>
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| - | <p>
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| - | - 0.5 ul of 100 uM reverse primer Stpx/PflI
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| - | </p>
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| - | <p>
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| - | - 25 ul of OneTaq (2x)
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| - | </p>
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| - | <p>
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| - | Touchdown PCR Protocol:
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| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
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| - | <tbody>
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| - | <tr>
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| - | <td valign="top">
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| - | <p>
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| - | Temp
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 64°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 68°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 57°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 68°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 72°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 4°C
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| - | </p>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td valign="top">
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| - | <p>
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| - | Time
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 3:00
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:15
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:30
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 2:20
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:15
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:30
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 2:20
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 5:00
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | Hold
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| - | </p>
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| - | </td>
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| - | </tr>
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| - | </tbody>
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| - | </table>
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| - | <p>
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| - | 15 cycles 15 cycles
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| - | </p>
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| - | <p>
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| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
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| - | </p>
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| - | <p>
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| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
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| - | </p>
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| - | <p>
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| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
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| - | </p>
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| - | <p>
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| - | <br/>
| |
| - | <img
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| - | width="549"
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| - | height="848"
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| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image002.jpg"
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| - | alt="https://lh4.googleusercontent.com/bqdSlOo8oD3UYMC71IhUNkVMtucJA86tKMLoyG8uzRaEsXeFPiG6KCmLF2Zzutp4YwFNAnDKumfVjQepHLHxwFDXDCzfmb6R6kEQZ6kWCokrwsIMmEXUPToQ"
| |
| - | />
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| - | </p>
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| - | <p>
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| - | Gel 1: From left to right. PflI, Ladder, Stpx.
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| - | </p>
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| - | <p>
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| - | <strong>8/7/13</strong>
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| - | </p>
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| - | <p>
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| - | Each PCR sample consists:
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| - | </p>
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| - | <p>
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| - | - 23 ul MilliQ water
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| - | </p>
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| - | <p>
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| - | - 1 ul of template (colony pick from CB15 culture in PYE)
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| - | </p>
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| - | <p>
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| - | - 0.5 ul of 100 uM forward primer Stpx/PflI
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| - | </p>
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| - | <p>
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| - | - 0.5 ul of 100 uM reverse primer Stpx/PflI
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| - | </p>
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| - | <p>
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| - | - 25 ul of OneTaq (2x)
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| - | </p>
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| - | <p>
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| - | Touchdown PCR Protocol:
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| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
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| - | <tbody>
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| - | <tr>
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| - | <td valign="top">
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| - | <p>
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| - | Temp
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 60°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 68°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 94°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 53°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 68°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 72°C
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 4°C
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| - | </p>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td valign="top">
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| - | <p>
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| - | Time
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 3:00
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:15
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:30
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 2:20
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 0:15
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| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
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| - | 0:30
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 2:20
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| - | </p>
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| - | </td>
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| - | <td valign="top">
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| - | <p>
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| - | 5:00
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| - | </p>
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| - | </td>
| |
| - | <td valign="top">
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| - | <p>
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| - | Hold
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| - | </p>
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| - | </td>
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| - | </tr>
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| - | </tbody>
| |
| - | </table>
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| - | <p>
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| - | 15 cycles 15 cycles
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| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
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| - | width="565"
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| - | height="659"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image003.gif"
| |
| - | alt="https://lh5.googleusercontent.com/DqUnHeJ07BhHQJ9_z-K1FUALQ9mJdtBbuipr0Uh7sOsGKwZ6mQMfPUhhKgPws_zNOklczf2gSwHh3I3jFfw2F8IYwIheS_H6vtPHzxoBAKd9kwHv_6txB1qJ"
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| - | />
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| - | </p>
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| - | <p>
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| - | Gel 2: From left to right. PflI, Stpx, Ladder.
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| - | </p>
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| - | <p>
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| - | <strong>8/8/13</strong>
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| - | </p>
| |
| - | <p>
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| - | Each PCR sample consists:
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| - | </p>
| |
| - | <p>
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| - | - 23 ul MilliQ water
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| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of template (colony pick from CB15 culture in PYE)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of 100 uM forward primer Stpx/PflI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of 100 uM reverse primer Stpx/PflI
| |
| - | </p>
| |
| - | <p>
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| - | - 25 ul of OneTaq (2x)
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| - | </p>
| |
| - | <p>
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| - | Touchdown PCR Protocol:
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| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
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| - | Temp
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| - | </p>
| |
| - | </td>
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| - | <td valign="top">
| |
| - | <p>
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| - | 94°C
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| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
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| - | 94°C
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| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
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| - | 56°C
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| - | </p>
| |
| - | </td>
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| - | <td valign="top">
| |
| - | <p>
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| - | 68°C
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| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
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| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="668"
| |
| - | height="707"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image004.jpg"
| |
| - | alt="https://lh3.googleusercontent.com/YilToZzn-PDtLPW8uFGD44m2k9IwTO1jCZqaYnZqaOQeYGFe35DDxBRTJ7LPkILMaoBUF96R3hlleWSA5PpGaey32VK5sSbXi48Asa6DPENg937Yc08Ahl3g"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 3: From left to right. PflI, Stpx, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/9/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Each PCR sample consists:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 23 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of template (colony pick from CB15 culture in PYE)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of 100 uM forward primer Stpx/PflI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of 100 uM reverse primer Stpx/PflI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of OneTaq (2x)
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="636"
| |
| - | height="680"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image005.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/PYYGKPLMPw1p6IphHGfuby6W346yi5YZQdWLw8snLI4j3qmwXUYSMtmi6ayJLoLllVRJMiMT7JTQtX6ctlp8b-OBPAz86VzXBjk_ef1nf_8bv4AzPb5DpHBU"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 4: From left to right. Ladder, Stpx, PflI.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/13/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Each PCR sample will contain:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 9.5 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of template (colony pick from CB15N colony pick in PYE)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer at 100 uM concentration Stpx/PflI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer at 100 uM concentration Stpx/PflI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12.5 ul of 2x Phusion with GC
| |
| - | </p>
| |
| - | <p>
| |
| - | PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 66°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="628"
| |
| - | height="731"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image006.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/nLvs_gSdKgYyAEDlUSGfCrv4cmw_KIH-0UFhWWzYTRs0fka-K-CGofDaKyJFtTP_KIQEPGVh-adD_g9o01j4EX8Z3zr8h9WGbbN6EGLCAwaDMwzEqcXDlTDK"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 5: From left to right. PflI, Stpx, Ladder, PflI, N/A, Stpx, N/A.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/15/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Two samples of Stpx and PflI were made.
| |
| - | </p>
| |
| - | <p>
| |
| - | Each PCR sample will contain:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 23 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of template (colony pick from CB15M colony pick in PYE)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of PflI/Stpx forward primers at 100 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul of PflI/Stpx reverse primers at 100 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 95°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 95°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 95°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:20
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="616"
| |
| - | height="700"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image007.jpg"
| |
| - | alt="https://lh5.googleusercontent.com/tzzRa988idhectW1gt9oCCjqz-4z6GNNdqNzaH8L-MrfWi_eDcAjTiecAadYSlQFArTzpsWsWBX5ECoZhtlF0w49xFwzleSoIJ6CxKdrlZ02V5IIJnAZlo7Q"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 6: From left to right. Stpx, PflI, Ladder, Stpx, PflI.
| |
| - | </p>
| |
| - | <p>
| |
| - | Stpx, PflI, TipF, DivJ PCR:
| |
| - | </p>
| |
| - | <p>
| |
| - | Four samples will contain:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer (Stpx, PflI, DivJ, TipF) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer (Stpx, PflI, DivJ, TipF) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N made from a colony pick diluted in water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <img
| |
| - | width="652"
| |
| - | height="696"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image008.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/SWGALEwjSN0ZCt3-nq8oCfO18MBZhUUs_kdvMYQkFKEVoqnGNppZK8HqPXMEtmyOqO9OrOH1g15-wM7pNSuMfTXNGZLV7HBnV9ScCDtZVMUD_4mYmcO64MjN"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 7: from left to right Stpx, PflI, TipF, DivJ, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/16/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Stpx and TipF PCR:
| |
| - | </p>
| |
| - | <p>
| |
| - | One Stpx sample and one TipF sample were made.
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul forward primer (Stpx/TipF) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul reverse primer (Stpx/TipF) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template from CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 9.5 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12.5 of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 120 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <img
| |
| - | width="676"
| |
| - | height="648"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image009.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/vYKcfML-noxqlCMJnj2czk-X_Ho7ydeUy_747PShlNAmkQl-e1wd_eNlQRfydrwTC0toqSp_Lchg3feW-9k1xsorRWJmB1YPsQe1aqkxl9GOOx8IajDFFC7L"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 8: left to right Stpx, ladder, TipF.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ran the remaining PCR product of TipF and DivJ (45 ul) on a 0.8% agarose gel made from 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60
| |
| - | minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 45 ul PCR product + 8 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="636"
| |
| - | height="833"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image010.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/cFyCO1F9O83Bz5bUsTU_dDZZ3CviKInNuugYn6AU5ZVIQLZ4D5J28EO9y75iOuQ54-ikl2_kmyz8G0ZlWQRsm9RUvgUzVJ93P7rQKzu9OAU20yzsjT28GGb4"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 9: from left to right TipF, DivJ, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>*gel shows bright bands at expected lengths of ~1.9 kb for TipF and ~600bp for divJ</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Purifying DNA from an Agarose Gel:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Cut band out in a dark room with a sterile razor and an UV lamp.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place the cut of gel band in a
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge the gel band for 10 minutes at 5,000 xg.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add five volumes of DNA Binding buffer to each volume of DNA sample.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Load the mixture into a Zymo-Spin Column in a collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Centrifuge at 10,000 xg for 30 seconds. Discard flow through.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 200 ul of DNA Wash buffer to the column and centrifuge at 10,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place the Zymo-Spin column into a new 1.5 ml tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Add 25 ul MilliQ water to column matrix and spin at 10,000 xg for 30 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/19/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | PflI and DivJ PCR:
| |
| - | </p>
| |
| - | <p>
| |
| - | Two samples:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul forward primer (PflI/DivJ) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul reverse primer (PflI/DivJ) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N made from a colony pick diluted in water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 9.5 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12.5 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:40
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:40
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="628"
| |
| - | height="1169"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image011.jpg"
| |
| - | alt="https://lh3.googleusercontent.com/w28lkbpORIACVZ9QTdPMfSiKE0KssCoP49fdhEpA37pofJPWkjeQm-SSnaSPaWxlouX_Y8MTcUzVjMvhYR8RckR-OELs5FFzlLSxE48byqxR21T7amwLFOe6"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 10: From left to right. PflI, ladder, DivJ.
| |
| - | </p>
| |
| - | <p>
| |
| - | Redo TipF and DivJ DNA Purification:
| |
| - | </p>
| |
| - | <p>
| |
| - | Four PCR samples (x2 TipF and x2 DivJ):
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer (TipF/DivJ) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer (TipF/DivJ) at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/20/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel of 8/19/13 Redo TipF and DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="627"
| |
| - | height="1031"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image012.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/ENrQ9lZxzVb3ZzqmkTc2UWbPA4qNQIitxVKToV6O-UVMUOnm91WBEBSj3ToOdlgm0QHusrqPG80JVZSFmTOXOrWfWdLpp5Cr2b-y9Z_1UnTG7gUwTXz8UEugtg"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 11: From left to right. TipF, DivJ, TipF, DivJ, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | Purifying DNA from an Agarose Gel:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Cut band out in a dark room with a sterile razor and an UV lamp.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place the cut of gel band in a
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge the gel band for 10 minutes at 5,000 xg.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add five volumes of DNA Binding buffer to each volume of DNA sample.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Load the mixture into a Zymo-Spin Column in a collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Centrifuge at 10,000 xg for 30 seconds. Discard flow through.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 200 ul of DNA Wash buffer to the column and centrifuge at 10,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place the Zymo-Spin column into a new 1.5 ml tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Add 25 ul MilliQ water to column matrix and spin at 10,000 xg for 30 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | 12 PCR Reactions of DivJ and TipF (6 each):
| |
| - | </p>
| |
| - | <p>
| |
| - | x6 TipF:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x6 DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <img
| |
| - | width="647"
| |
| - | height="388"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image013.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/190JIiQvmde5rQQqCHKOnGtsy1Wwguah8cUSG7_Aoto4180iVEomRFnf-BEbm8pmKKLK3buW4br9Lkw2Kp0PAkx90_XC1AkmRaBB6umAo6Ayn_vnBGAlqQUh"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 12: From left to right. TipF, TipF, TipF, TipF, TipF, TipF, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="658"
| |
| - | height="360"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image014.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/rAiWnkYRTJvwjt5qQY8V4m73hklSPQHVkFqc37iQn4tzQTlVFJIFfSnDi0h0BzfnoWIpmQrylNNIDh9XXOdfnjIKWLKFf7g-_WrshQGXU_KB6LDlgTIh-LFp"
| |
| - | />
| |
| - | Gel 13: From left to right. DivJ, DivJ, DivJ, DivJ, DivJ, DivJ, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/21/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Checking the Purified Samples of TipF and DivJ for One Band:
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 5 ul PCR product + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="673"
| |
| - | height="612"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image015.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/aVq4j7WQqbwkezTZN08FKVk5Z9pohk1EPdwUaMAdJe0aDIOfOTWaBCkehMM9C6TNttXPMm3ETPhNZwq5O78JxsCk2WuzEiW-muVVIT0a7rhfo5H2UfwEvbeu"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 14: From left to right. TipF, DivJ, Ladder.
| |
| - | </p>
| |
| - | <p>
| |
| - | x2 TipF:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x2 DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | *Forgot to take a picture of the gel but the gel picture was not good. The TipF samples were not near the expected size. The DivJ samples were smeared.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/22/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Restriction Digest with NdeI and NsiI
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | TipF
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | pVCPFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 28 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 35 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 3.1 10x buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 3.1 10x buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 3.1 10x buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 3.1 10x buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NsiI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NsiI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NsiI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NsiI
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 15 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 8 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 38 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 38 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 1) Incubate in the PCR thermocycler at 37°C for 30 minutes then at 65°C for 20 minutes to inactivate.
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="648"
| |
| - | height="571"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image016.jpg"
| |
| - | alt="https://lh3.googleusercontent.com/OVx2lPgFKvJ1QRg5gulbeMNwgOMzo9anTDfxXH6AKvdAG2cPqraSmWFGpjay5F00DQ-BphAhrydJvWfNAIN8xOfNyxYQqhlpfX5OUhX0qRc43lp-eu9FNV8o"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 15: From left to right. Ladder, DivJ with NotI, DivJ without NotI, pXYFPC-2, pVCPFPC-4.
| |
| - | </p>
| |
| - | <p>
| |
| - | Purifying Vector DNA from an agarose gel
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Cut band out in a dark room with a sterile razor and an UV lamp.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place the cut of gel band in an agarose spin column.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge the gel band for 10 minutes at 5,000 xg.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add five volumes of DNA Binding buffer to each volume of DNA sample.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Load the mixture into a Zymo-Spin Column in a collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Centrifuge at 10,000 xg for 30 seconds. Discard flow through.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 200 ul of DNA Wash buffer to the column and centrifuge at 10,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place the Zymo-Spin column into a new 1.5 ml tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Add 25 ul MilliQ water to column matrix and spin at 10,000 xg for 30 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | NotI and DivJ Restriction Digest:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12.5 ul DivJ DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul CutSmart 10X buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul NotI
| |
| - | </p>
| |
| - | <p>
| |
| - | - 31.5ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Incubate in the PCR thermocycler at 37°C for 30 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Refer to Gel 15.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/23/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | DivJ and TipF Ligations
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix for pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 12 ul of pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 12 ul of pVCPFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3 ul of 10x T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3 ul of 10x T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1.5 ul T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1.5 ul T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Six total ligation reactions:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of TipF
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 6) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of TipF
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Incubate ligation reactions for 30 minutes or greater at room temperature.
| |
| - | </p>
| |
| - | <p>
| |
| - | Heat Shock Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 40 ul of Top10 Chemicompetent cells in 10 ul of each ligation reaction.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place in a 42°C water bath for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Place back on ice for 5 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add 200 ul of LB to a microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Transfer the Top10 Chemicompetent cells and ligation reaction to the microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Incubate for one hour at 37°C at 200 rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | Plating the Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Obtain three plates of LB and gentamicin and three plates of LB and kanamycin.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Warm up the plates in the 37°C incubator prior to plating.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Pipet all of the transformation into the plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Using a bunsen burner, heat up the steel rod.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Create streaks with the steel rod.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Wait for 5 minutes until all the liquid is soaked up by the plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Tape up the plates and/or place the plates in a plastic bag.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place plates in an incubator at 37°C.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/25/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Created an overnight of the DivJ and TipF plates by picking five colonies from each LB and gentamicin plate plus one colony from the negative control. The
| |
| - | colony picks were diluted in 20 ul of MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/26/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Purpose</strong>
| |
| - | : colony plasmid isolation
| |
| - | </p>
| |
| - | <p>
| |
| - | Miniprep of TipF and DIvJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of 7X Lysis buffer to 600 ul of <em>e. coli</em> culture in a 1.5 ml microcentrifuge tube. Mix by inverting the tube 4-6 times and lyse
| |
| - | samples for 1-2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Add 350 ul of cold Neutralization buffer. Mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge at 16,000 xg for 2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Transfer the supernatant into the Zymo-Spin IIN column .
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Place column into a collection tube and centrifuge at 11,000 xg for 15 seconds. Discard the flow through and place the column back into the same
| |
| - | collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Add 200 ul of ENdo Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 400 ul of Zyppy Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 ul of MilliQ water. Let stand for one minute at room temperature. Centrifuge at
| |
| - | 11,000 xg for 15 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Purpose: </strong>
| |
| - | diagnostic PCR reactions to amplify target sequences
| |
| - | </p>
| |
| - | <p>
| |
| - | PCR:
| |
| - | </p>
| |
| - | <p>
| |
| - | x5 TipF
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of eluted TipF DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x5 DivJ
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of eluted DivJ DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x2 Positive Control
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer DivJ/TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer DivJ/TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x2 Negative Control
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of colony pick from the negative control plate
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer DivJ/TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer DivJ/TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.46g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <img
| |
| - | width="663"
| |
| - | height="627"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image017.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/4X7v4TWdIxeDrp2id7tHK7m0I-APzihgsU4ZmWyk90kDvjuXBWaQp2u2RrFRpZoPkYkrb-cwadbKNSxt5CNKj7LU4IyoHruJLCRdT0i5tPrawe2_37jRc1Oz"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 16: From left to right: Ladder, TipF1, TipF2, TipF3, TipF4, TipF5, Negative Control from TipF plate, Positive Control, DivJ1, DivJ2, DivJ3, DivJ4,
| |
| - | DivJ5, Negative Control from DivJ plate, Positive. Control.
| |
| - | </p>
| |
| - | <p>
| |
| - | Sequencing:
| |
| - | </p>
| |
| - | <p>
| |
| - | We sent in TipF5 and DivJ3 and DivJ4 in for sequencing.
| |
| - | </p>
| |
| - | <p>
| |
| - | For TipF5, we used 10 ul of the miniprepped TipF DNA and 3 ul of the forward primer at 1 uM concentration. Also 10 ul of the miniprepped TipF DNA and 3 ul
| |
| - | of the reverse primer at 1 uM concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | For DivJ3, we used 10 ul of the miniprepped DivJ DNA and 3 ul of the forward primer at 1 uM concentration. Also 10 ul of the miniprepped DivJ DNA and 3 ul
| |
| - | of the reverse primer at 1 uM concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | For DivJ4, we used 10 ul of the miniprepped DivJ DNA and 3 ul of the forward primer at 1 uM concentration. Also 10 ul of the miniprepped DivJ DNA and 3 ul
| |
| - | of the reverse primer at 1 uM concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/27/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | x10 TipF samples:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of eluted miniprepped TipF DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer TipF at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | x10 DivJ samples:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of eluted miniprepped DivJ DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 56°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 49°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="662"
| |
| - | height="521"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image018.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/hhaySBlFVhPr1rNS91eWcAMKFny5eg0-gclhDFyptP2Wqx2dV-ZY2fXGLChwSBYHKoec_DhkaGhKH58hPgfx3GGDIRbYp_i5Yu_lKuBuaZhrRsDEFi4G0ZJR"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 17: From left to right. Ladder, TipF1, TipF2, TipF3, TipF4, TipF5,TipF6, TipF7, TipF8, TipF9, TipF10, Negative Control from TipF plate, Positive
| |
| - | Control.
| |
| - | </p>
| |
| - | <p>
| |
| - | *TipF3 shows a band at expected length
| |
| - | <img
| |
| - | width="669"
| |
| - | height="549"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image019.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/7ppxz3DGvGHqHvh98b0xUbtiKhMRuGF9I5TbpynN7trvvHWHcRWWWb0AMe6EPg-TrvGdLttAOEO_UwXdLjcjM2THc-j3gfW9oVB2uH1HL9dH5_D8XwzU_927"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 18: From left to right. Ladder, DivJ1, DivJ2, DivJ3, DivJ4, DivJ5, DivJ6, DivJ7, DivJ8, DivJ9, DivJ10, Negative Control from DivJ plate, Positive.
| |
| - | Control.
| |
| - | </p>
| |
| - | <p>
| |
| - | Overnight culture of TipF3 for Miniprep:
| |
| - | </p>
| |
| - | <p>
| |
| - | From the colony pick from the LB + gentamicin TipF plate, we diluted it in 20 ul of MilliQ water. That mixture was then transferred into 4 ml of LB. 6 ul
| |
| - | of gentamicin antibiotic was added. The overnight culture was stored in the incubator at 28°C at 220 rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/28/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Miniprep of Overnight TipF3 Culture:
| |
| - | </p>
| |
| - | <p>
| |
| - | Spin down overnight culture for 10 minutes at 3,000 xg. Discard 3.4 ml of the supernatant. Resuspend the pellet.
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of 7X Lysis buffer to 600 ul of <em>e. coli</em> culture in a 1.5 ml microcentrifuge tube. Mix by inverting the tube 4-6 times and lyse
| |
| - | samples for 1-2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Add 350 ul of cold Neutralization buffer. Mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge at 16,000 xg for 2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Transfer the supernatant into the Zymo-Spin IIN column .
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Place column into a collection tube and centrifuge at 11,000 xg for 15 seconds. Discard the flow through and place the column back into the same
| |
| - | collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Add 200 ul of ENdo Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 400 ul of Zyppy Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 ul of MilliQ water. Let stand for one minute at room temperature. Centrifuge at
| |
| - | 11,000 xg for 15 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | Sequencing of TipF3:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 10 ul of miniprepped TipF + 3 ul of TipF forward primer at 1 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 10 ul of miniprepped TipF + 3 ul of TipF reverse primer at 1 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/29/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | pVan-for and eGYC-1 PCR of TipF Colony Picks:
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 60 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | - 24 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul pVan-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Colony Picks:
| |
| - | </p>
| |
| - | <p>
| |
| - | - Colony pick of TipF in the LB and gentamicin plate into 20 ul of MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Twelve PCR Reactions:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | TipF 1-10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Negative Control
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Positive Control
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of colony pick in water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of Colony pick in water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of pYCFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 60°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 53°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.46g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="648"
| |
| - | height="612"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image020.jpg"
| |
| - | alt="https://lh3.googleusercontent.com/_IXuUdIxH-rNGplWsIcisY0rwX5Df_tdURpkRPxzhKJci6Qa8T-eXaCc_kKupss_soxmwBqr_IP9ubzOFghide3mWiBaM7gKLRD_3UPf27ShZu1GJz40XV9t"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 19: From left to right: Ladder, Gel Protocol: We created an 0.8% agarose gel by obtaining 0.4g of agarose and 50 ml of 1X TBE buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/30/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | x2 DivJ samples:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul template of CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | - 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Negative Control:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 20 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul forward primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 ul reverse primer DivJ at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul of 2x Phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 64°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 57°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="615"
| |
| - | height="767"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image021.jpg"
| |
| - | alt="https://lh5.googleusercontent.com/LDVwWsEioeGss3CV3LvE7md0zY9JiBCF-y6s_tR_6YCz-tOKsMNN_8ks9_ddvq1wWIAcuQrC8gp_WvAEouJg_PmBVAg8HW2CoRJwc6aBjlmYmstF98Jbv_E_"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 20: From left to right. Ladder, DivJ1, Remaining DivJ1, DivJ2.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>8/31/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Miniprep of TipF.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/1/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.4g of agarose and 50 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="679"
| |
| - | height="500"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image022.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/tK8oZgdG2FU87eoxdPw8ZEKBLKUpuCdSnqqWc-po9S3_RfTKVEWcdnVTBQ6IWQtHp62_RGiwwZDGqYJtzhkUhKMGcXVHLpi-Djy8VxxQztYAmPcFIU7k8HGV"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 22: From left to right. TipF, TipF Ladder.
| |
| - | <img
| |
| - | width="558"
| |
| - | height="515"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image023.jpg"
| |
| - | alt="https://lh3.googleusercontent.com/yb0Bb8EiMtYFdZuwckFMfJI1F-LcIUm0A0PGK4FI613yk9IOXoVjRA31l7C66MNvxHfDSk1w1tb-hwxQNa0ksC-b2olDgqt-mCPLvco7hgtGhquuNCv0cu6U"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 23: From left to right. Ladder, Vector C, Vector Y.
| |
| - | </p>
| |
| - | <p>
| |
| - | Cut out the vectors and purify along with DivJ.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ligation:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix for pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 7 ul Vector C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 7 ul Vector Y
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2 ul of 10x T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2 ul of 10x T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Four total ligation reactions:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1) pXYFPC-2 Mastermix + 5 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3) pVCPFPC-4 + 5 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2) pXYFPC-2 Mastermix + 326 DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4) pVCPFPC-4 + 326 DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Heat Shock Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of BL21 Chemicompetent cells in 10 ul of each ligation reaction.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place in a 42°C water bath for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Place back on ice for 5 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add 200 ul of LB to a microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Transfer the BL21 Chemicompetent cells and ligation reaction to the microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Incubate for one hour at 37°C at 200 rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | Plating the Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Obtain two plates of LB and gentamicin and three plates of LB and kanamycin.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Warm up the plates in the 37°C incubator prior to plating.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Pipet all of the transformation into the plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add glass beads to the plates and shake.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Remove the glass beads and incubate overnight at 37°C.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/2/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | PCR of the 326 DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | Same protocol as 8/30/13
| |
| - | </p>
| |
| - | <p>
| |
| - | Purified 326 DivJ by cutting it out of the gel and doing a clean and concentrate.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/3/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Transformation #3</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Purpose: </strong>
| |
| - | To insert DivJ into Vectors and transform ligated fusion vectors/DivJ into competent cells.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Work Flow:</strong>
| |
| - | Restrict plasmids Y and C. Run restricted plasmids and DivJ(PCR product) on agarose gel. Cut out DNA/ Clean DNA/ Concentrate DNA. Restriction digest of
| |
| - | DivJ. Clean and Concentrate DivJ. Ligate sequences. Transform into electrocompetent cells (e.coli top ten).
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Restrict plasmids</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Vector C</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 uL DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 uL CutSmart (10x) buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 uL NdeI
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 uL AclI
| |
| - | </p>
| |
| - | <p>
| |
| - | 38 uL MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Vector Y</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 uL DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 uL CutSmart (10x) buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 uL NdeI
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 uL AclI
| |
| - | </p>
| |
| - | <p>
| |
| - | 38 uL MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | -incubate at 37° C for 30 min.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Agarose Gel</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 0.8% 50ml agarose gel
| |
| - | <img
| |
| - | width="663"
| |
| - | height="559"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image024.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/8Z94dLuN_TZ8CpzxsA25ET2e0SKR37eUQz8XUO0J0BNbbULYtojVfaTZopCKT3WBUMftGSQfGDUV7lzpKYanxDRTbH4NTsRDZmadzs7ewraJ8LdXxfX_lAKw"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 24: From left to right. 326 DivJ, Vector Y, Vector C.
| |
| - | </p>
| |
| - | <p>
| |
| - | -cut bands out of gel
| |
| - | </p>
| |
| - | <p>
| |
| - | -clean/concentrate
| |
| - | </p>
| |
| - | <p>
| |
| - | -elute vectors to 12 ul
| |
| - | </p>
| |
| - | <p>
| |
| - | -elute DivJ to 30 ul
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Restriction Digest (DivJ)</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | 30 ul DivJ DNA
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 ul CutSmart (10x) buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul AclI
| |
| - | </p>
| |
| - | <p>
| |
| - | 13 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | -incubate at 37° C for 30 min.
| |
| - | </p>
| |
| - | <p>
| |
| - | -clean/concentrate
| |
| - | </p>
| |
| - | <p>
| |
| - | -elute to 10 ul
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Ligation reactions</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Vec C master mix (MMC) Vec Y master mix (MMY)</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 7 ul vector C 7 ul vector Y
| |
| - | </p>
| |
| - | <p>
| |
| - | 2 ul T4 buffer 2 ul T4 buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul T4 ligase 1 ul T4 ligase
| |
| - | </p>
| |
| - | <p>
| |
| - | -prepare reactions
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Neg Control C (NC) Vec C w/ DivJ insert (CD)</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 ul MMC 5 ul MMC
| |
| - | </p>
| |
| - | <p>
| |
| - | 5ul MilliQ H2O 5 ul DivJ
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>Neg Control Y (NY) Vec Y w/ DivJ insert (NC)</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 ul MMY 5 ul MMY
| |
| - | </p>
| |
| - | <p>
| |
| - | 5 ul MilliQ H2O 5 ul DivJ
| |
| - | </p>
| |
| - | <p>
| |
| - | - incubate at room temp for 30 min.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Electroporate</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Top10 electrocompetent cells
| |
| - | </p>
| |
| - | <p>
| |
| - | -for each ligation product
| |
| - | </p>
| |
| - | <p>
| |
| - | 250 ul top 10 electrocompetent cells
| |
| - | </p>
| |
| - | <p>
| |
| - | 10 ul ligation reaction
| |
| - | </p>
| |
| - | <p>
| |
| - | *note too much salt
| |
| - | </p>
| |
| - | <p>
| |
| - | -recovery in 1 ml LB @ 37° C 1 hour
| |
| - | </p>
| |
| - | <p>
| |
| - | -plate on LB/kan plates for Y vectors and LB/gen plates for C vectors
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/4-5/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Re-did the 326 DivJ since plates did not look good. Same procedure except we now used BL21 electrocompetent cells.
| |
| - | </p>
| |
| - | <p>
| |
| - | Also synthesized the minimal DivJ by:
| |
| - | </p>
| |
| - | <p>
| |
| - | Creating an oligo mix consisting of 5 ul of each oligo at 100 uM concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | Synthesize PCR protocol:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of oligo mix
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul forward primer at 100 uM concentration (NdeI)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul reverse primer at 100 uM concentration (AciI)
| |
| - | <br/>
| |
| - | - 23 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul 2X phusion mix with GC buffer
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 63°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Amplify PCR Protocol:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 1 ul of the synthesis PCR product
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul forward primer at 100 uM concentration (NdeI)
| |
| - | </p>
| |
| - | <p>
| |
| - | - 0.5 ul reverse primer at 100 uM concentration (AciI)
| |
| - | <br/>
| |
| - | - 23 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 25 ul 2X phusion mix with GC buffer
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 63°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.4g of agarose and 50 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="578"
| |
| - | height="636"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image025.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/L2a_H3ORYYxE0YrX_pvS4mcGcpSBn9f7Dz3JVYjM5DKDHOJYROMHOU2KO9bHnudOnTzSeiq5aA_jIgFKaVsOokDAu4QEuuriEvYTXiXv2TUOEvqfyoga8r8E"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 25: From left to right. Ladder, Synth PCR, Amp PCR, DivJ, Vector C digested, Vector Y digested.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/6/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Electroporation but this time with BL21 electrocompetent cells.
| |
| - | </p>
| |
| - | <p>
| |
| - | Plates from this did not look good as well.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>* note BL21 was not a good choice for transformations due to the presence of RecA gene.</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/7/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Purpose: </strong>
| |
| - | prepare top 10 chemi competent cells for use in future transformations.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>
| |
| - | * note bad results from transformation attempts with top ten electrocompetent cells may have been due to salty ligations and low yields. The chemi
| |
| - | competent cells may allow us to use more of our ligation products during transformations.
| |
| - | </strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Making Top10 Chemicompetent Cells:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Grow Top10 SOB overnight between the temperatures 20°C and 33°C.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Measure the OD and bring the OD back to 0.1.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Add 2.5 ml of Top10 SOB to SOB.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Incubate at 31°C for one hour and 20 minutes or until the OD is 0.6.
| |
| - | <br/>
| |
| - | 5) Create 5 ml of 1X Washing buffer: 2.5 ml of Dilution buffer and 2.5 ml of Wash buffer (2X)
| |
| - | <br/>
| |
| - | 6) Create 5 ml of 1X Competent buffer: 2.5 ml Dilution buffer and 2.5 ml of Competent buffer (2X)
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Incubate the culture on ice for ten minutes and then pellet cells at 2,500 xg for ten minutes at 0-4°C.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Remove supernatant. Gently resuspend cells in 5 ml of ice cold 1X Wash buffer. Re-pellet as in step 7.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Remove supernatant completely. Gently resuspend cells in 5 ml of ice cold 1X Competent buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | 10) Aliquot 100-200 ul. Flash freeze.
| |
| - | </p>
| |
| - | <p>
| |
| - | 11) Store in -80°C freezer.
| |
| - | </p>
| |
| - | <p>
| |
| - | Making LB+Kanamycin and LB+Gentamicin Agar Plates:
| |
| - | </p>
| |
| - | <p>
| |
| - | To make a 500 ml batch:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 475 ml of MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 g of Tryptone
| |
| - | </p>
| |
| - | <p>
| |
| - | - 5 g of NaCl
| |
| - | </p>
| |
| - | <p>
| |
| - | - 2.5 g of yeast Extract
| |
| - | </p>
| |
| - | <p>
| |
| - | - 7.5 g of Agar
| |
| - | </p>
| |
| - | <p>
| |
| - | We made x2 500 ml batches.
| |
| - | </p>
| |
| - | <p>
| |
| - | Combine the reagents and shake until the solutes have dissolved. Adjust the ph to 7.0. Sterilize by autoclaving for 20 minutes on liquid cycle.
| |
| - | </p>
| |
| - | <p>
| |
| - | Take out of the autoclave and let the solution cool. Place in a water bath to prevent solid formation.
| |
| - | </p>
| |
| - | <p>
| |
| - | Add 1.25 ml of gentamicin antibiotic to one of the 500 ml batches and 500 ul of kanamycin to the other batch.
| |
| - | </p>
| |
| - | <p>
| |
| - | Pour into petri dishes about half way. Let agar solidify. Place in the refrigerator.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/8/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Testing the Top10 Chemicompetent Cells:
| |
| - | </p>
| |
| - | <p>
| |
| - | We tested our Top10 Chemicompetent cells by using different amount of DNA. We used 300 ng of DNA and 30 ng of DNA from our vec C and Vec Y plasmid stocks.
| |
| - | The 300 ng of DNA plates had more growth than the 30 ng of DNA plates.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>Transformations</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>K1 G1</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul dilute vector Y 1 ul dilute vector C
| |
| - | </p>
| |
| - | <p>
| |
| - | 100 ul CC top 10 cells 100 ul CC top 10 cells
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>K2 G2</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul Vector Y 1 ul Vector C
| |
| - | </p>
| |
| - | <p>
| |
| - | 100 ul CC top 10 cells 100 ul CC top 10 cells
| |
| - | </p>
| |
| - | <p>
| |
| - | <u>KN GN</u>
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul MilliQ H2O 1 ul MilliQ H2O
| |
| - | </p>
| |
| - | <p>
| |
| - | 100 ul CC top 10 cells 100 ul CC top 10 cells
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/9/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Top10 Chemi competent Cell Transformation of DivJ
| |
| - | </p>
| |
| - | <p>
| |
| - | Heat Shock Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of Top10 Chemicompetent cells in 10 ul of each ligation reaction.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place in a 42°C water bath for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Place back on ice for 5 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add 500 ul of SOC to a microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Transfer the Top10 Chemicompetent cells and ligation reaction to the microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Incubate for one hour at 37°C at 200 rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | Colony PCR of TipF
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 60 ul of 2x OneTaq
| |
| - | </p>
| |
| - | <p>
| |
| - | - 24 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul new-pVan-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul new-eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Colony Picks:
| |
| - | </p>
| |
| - | <p>
| |
| - | - Colony pick of TipF in the LB and gentamicin plate into 20 ul of MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Twelve PCR Reactions:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | TipF 1-10
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Negative Control
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Positive Control
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 9 ul of Mastermix
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of colony pick in water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of Colony pick in water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul of pYCFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 60°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 53°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="623"
| |
| - | height="647"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image026.jpg"
| |
| - | alt="https://lh4.googleusercontent.com/GNkoIGL6ey6G3qWarAJ6T9LCnJakwC0g_25gQbE6GfJSFi4OarBnpOylrOifrVAX5CWfW6pXu6YYCb8MxPKeXz8NQeP2H9sg6nBRUnJacGo1Jk7ieFjYTYet"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 26: From left to right. Ladder, TipF1, TipF2, TipF3, TipF4, TipF5,TipF6, TipF7, TipF8, TipF9, TipF10, Negative Control from TipF plate, Positive
| |
| - | Control.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/10/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.8g of agarose and 100 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="424"
| |
| - | height="817"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image027.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/8KLJ_O2uwWLh26kx0J9BmN1q3cUhzDOqO3BVG-LTZSm5oTgbABH3jeG_Ede7CKW_vseeL0D3foeiRBeKF6D9hmPA5f8lGiM9mqh7Eejju_aepZwGtr5dm5m0"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 27: From left to right. Top: Ladder, T1, T2, T3, T4, T5, T6, T7, Y8, T9, T10, CD1, CD2, CD3, CD4, CD5, CD6, CD7, NC, PC. Botton: Ladder, CD8, CA1, CA2,
| |
| - | CA3, CA4, CA5, CA6, CA7, CA8, CA9, CA10, NC, PC, YD1, YD2, YD3, YD4, NY, PY. T = TipF, CD = 326 DivJ in pVCPFPC-4. CA = Minimal DivJ in pVCPFPC-4. YD = 326
| |
| - | DivJ in pXYFPC-2. NC = Negative control in pVCPFPC-4, PC = Positive control in pVCPFPC-4, NY = negative control in pXYFPC-2, PY = positive control in
| |
| - | pXYFPC-2.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/11/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | PCR of 326 DivJ and Minimal DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | 326 DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul template from CB15N glycerol stock
| |
| - | </p>
| |
| - | <p>
| |
| - | 2.5 ul DivJ forward primer at 10 uM concentration (NdeI)
| |
| - | </p>
| |
| - | <p>
| |
| - | 2.5 ul DivJ reverse primer at 10 uM concentration (AciI)
| |
| - | </p>
| |
| - | <p>
| |
| - | 19 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | 25 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Minimal DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1 ul of Synthesis PCR product
| |
| - | </p>
| |
| - | <p>
| |
| - | 0.5 ul DivJ forward primer at 100 uM concentration (248)
| |
| - | </p>
| |
| - | <p>
| |
| - | 0.5 ul DivJ reverse primer at 100 uM concentration (328)
| |
| - | </p>
| |
| - | <p>
| |
| - | 23 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | 25 ul of 2x phusion mix with GC buffer
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 64°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 98°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 57°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 72°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.4g of agarose and 50 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="491"
| |
| - | height="681"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image028.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/a0iVc1mVbRtzYgBDcDj5eBnYCT3-53XJeQ309xFrJm3EEJjrLJ9YhDCs-r0I-aAwcY_xaU9tkSIIZZ_shodrCT3EzzpuBCur96dD_q3DwC0tjDRgvHAylI3v"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 28: From left to right. Ladder, 326 DivJ, Minimal DivJ, pXYFPC-2, pYCFPC-4.
| |
| - | </p>
| |
| - | <p>
| |
| - | -start overnight cultures CA
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/12/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Miniprep of Overnight CA8, CA9, YD1 and YD3 Cultures:
| |
| - | </p>
| |
| - | <p>
| |
| - | Spin down overnight culture for 10 minutes at 3,000 xg. Discard 3.4 ml of the supernatant. Resuspend the pellet.
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of 7X Lysis buffer to 600 ul of <em>e. coli</em> culture in a 1.5 ml microcentrifuge tube. Mix by inverting the tube 4-6 times and lyse
| |
| - | samples for 1-2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Add 350 ul of cold Neutralization buffer. Mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge at 16,000 xg for 2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Transfer the supernatant into the Zymo-Spin IIN column .
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Place column into a collection tube and centrifuge at 11,000 xg for 15 seconds. Discard the flow through and place the column back into the same
| |
| - | collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Add 200 ul of Endo Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 400 ul of Zyppy Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 ul of MilliQ water. Let stand for one minute at room temperature. Centrifuge at
| |
| - | 11,000 xg for 15 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | Purification of DivJ:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Cut band out in a dark room with a sterile razor and an UV lamp.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place the cut of gel band in a
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge the gel band for 10 minutes at 5,000 g.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add five volumes of DNA Binding buffer to each volume of DNA sample.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Load the mixture into a Zymo-Spin Column in a collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Centrifuge at 10,000 g for 30 seconds. Discard flow through.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 200 ul of DNA Wash buffer to the column and centrifuge at 10,000 g for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place the Zymo-Spin column into a new 1.5 ml tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 9) Add 12 ul MilliQ water to column matrix and spin at 10,000 g for 30 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | Restriction Digest of DivJ:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Minimal DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 326 DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | pYCFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 28 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 28 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3.5 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3.5 ul DNA
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 10X CutSmart buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 10X CutSmart buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 10X CutSmart buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5 ul 10X CutSmart buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul AciI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul AciI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul AciI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul AciI
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1 ul NdeI
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 15 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 15 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 39.5 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 39.5 ul MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Incubate at 37°C for 30 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.4g of agarose and 50 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 50 ul PCR product + 10 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="616"
| |
| - | height="624"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image029.jpg"
| |
| - | alt="https://lh5.googleusercontent.com/XB8Aq3qh47QAm_pbu5Jw0zhlWyNxLLyfUD5o2bf0C8axJ7Yjdo1OUM-okLuMvpCKjyyAFy4ulxJPycne0T_xvXIR9bGYcdwczwkl1yRLZ4DVH0VzdjoSjq_O"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 29: From left ro right. Ladder, pXYFPC-2, pYCFPC-4.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/13/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Clean and concentrated DivJ and eluted it to 12 ul. Cut out the vectors. Clean and concentrated and eluted it to 15 ul each.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ligation of DivJ:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix of pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Mastermix of pYCFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 12 ul pXYFPC-2
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 12 ul pYCFPC-4
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3 ul 10X T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3 ul 10X T4 buffer
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1.5 ul T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1.5 ul T4 Ligase
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Six total ligation reactions:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 1) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of 326 DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of 326 DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 2) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of MilliQ water
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 3) 5.5 ul of Mastermix for pXYFPC-2 + 4.5 ul of Minimal DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 6) 5.5 ul of Mastermix for pVCPFPC-4
| |
| - | </p>
| |
| - | <p>
| |
| - | + 4.5 ul of Minimal DivJ
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | Incubate ligation reactions for 30 minutes or greater at room temperature.
| |
| - | </p>
| |
| - | <p>
| |
| - | Heat Shock Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of Top10 Chemicompetent cells in 1-2 ul of each ligation reaction.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Place in a 42°C water bath for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Place back on ice for 5 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Add 500 ul of SOC to a microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Transfer the Top10 Chemicompetent cells and ligation reaction to the microcentrifuge tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Incubate for one hour at 37°C at 200 rpm.
| |
| - | </p>
| |
| - | <p>
| |
| - | Plating the Transformations:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Obtain three plates of LB and gentamicin and three plates of LB and kanamycin.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Warm up the plates in the 37°C prior to plating.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Pipet all of the transformation into the plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Using a bunsen burner, heat up the steel rod.
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Create streaks with the steel rod.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Wait for 5 minutes until all the liquid is soaked up by the plate.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Tape up the plates and/or place the plates in a plastic bag.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Place plates in an incubator at 37°C.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/14/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Colony PCR of TipF and DivJ Plates:
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix for pVCPFPC-4 plasmids:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 110 ul 2x OneTaq
| |
| - | </p>
| |
| - | <p>
| |
| - | - 44 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 22 ul Pvan-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 22 ul eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix for pXYFPC-2 plasmids:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 55 ul 2x OneTaq
| |
| - | </p>
| |
| - | <p>
| |
| - | - 22 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 11 ul Pxyl-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 11 ul eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel Protocol: We created an 0.8% agarose gel by obtaining 0.48g of agarose and 60 ml of 1X TBE buffer. Run at 100 volts for 60 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | Ladder consists of 5 ul DNA Standard + 2 ul 6X gel loading buffer + 5 ul MilliQ water.
| |
| - | </p>
| |
| - | <p>
| |
| - | Samples consist of 10 ul PCR product + 2 ul 6X gel loading buffer.
| |
| - | </p>
| |
| - | <p>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <img
| |
| - | width="600"
| |
| - | height="742"
| |
| - | src="file:///C:/Users/Kevin/AppData/Local/Temp/msohtmlclip1/01/clip_image030.jpg"
| |
| - | alt="https://lh6.googleusercontent.com/n5LciA8J7UC-oHxlEgkvUaCu0LrRc-kryMfW6kyvxCi6dxLbcpsGurCfCEm2KCS2xJNNjMUHuHBBV1cQlDVtE1Oqf4C2zlTtYj7IZJsiPvUwPgw-9nxLkoq6"
| |
| - | />
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 30: From left to right. Top: CD1, CD2, CD3, CD4,CD5, CD6, CD7, CD8, CD9, CD10 CA1, CA2, CA3, CA4, CA5, CA6, CA7, CA8, Positive Control for pVCPFPC-4.
| |
| - | Bottom: CA10, Positive Control for pVCPFPC-4, YA1, YA2, YA3, YA4, YA5, YA6, YA7, YA8. YA9, YA10, Positive Control for pXYFPC-2. CD = 326 DivJ in pVCPFPC-4.
| |
| - | CA = Minimal DivJ in pVCPFPC-4. YA = Minimal DivJ in pXYFPC-2.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/15/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Overnight Cultures of YA1, YA2, YA3, YA4, YA5, CA5, CA10, CD2, CD3:
| |
| - | </p>
| |
| - | <p>
| |
| - | For each overnight:
| |
| - | </p>
| |
| - | <p>
| |
| - | 3 ml of SOC. For pVCPFPC-4, add in 4.5 ul of gentamicin. For pXYFPC-2, add in 1.8 ul of kanamycin.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/16/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Miniprep of Overnight Cultures:
| |
| - | </p>
| |
| - | <p>
| |
| - | 1) Add 100 ul of 7X Lysis buffer to 600 ul of <em>e. coli</em> culture in a 1.5 ml microcentrifuge tube. Mix by inverting the tube 4-6 times and lyse
| |
| - | samples for 1-2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 2) Add 350 ul of cold Neutralization buffer. Mix thoroughly. Neutralization is complete when sample becomes yellow and precipitate has formed.
| |
| - | </p>
| |
| - | <p>
| |
| - | 3) Centrifuge at 16,000 xg for 2 minutes.
| |
| - | </p>
| |
| - | <p>
| |
| - | 4) Transfer the supernatant into the Zymo-Spin IIN column .
| |
| - | </p>
| |
| - | <p>
| |
| - | 5) Place column into a collection tube and centrifuge at 11,000 xg for 15 seconds. Discard the flow through and place the column back into the same
| |
| - | collection tube.
| |
| - | </p>
| |
| - | <p>
| |
| - | 6) Add 200 ul of ENdo Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 7) Add 400 ul of Zyppy Wash buffer to the column. Centrifuge at 11,000 xg for 30 seconds.
| |
| - | </p>
| |
| - | <p>
| |
| - | 8) Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 ul of MilliQ water. Let stand for one minute at room temperature. Centrifuge at
| |
| - | 11,000 xg for 15 seconds to elute the DNA.
| |
| - | </p>
| |
| - | <p>
| |
| - | Nine Sequencing Samples:
| |
| - | </p>
| |
| - | <p>
| |
| - | YA1, YA2, YA3, YA4, YA5, CA5, CA10, CD2, CD3
| |
| - | </p>
| |
| - | <p>
| |
| - | 10 ul of miniprepped DNA plasmid with 3 ul of the resepective forward/reverse primer at 1 uM concentration.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/17/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Made more gentamicin and kanamycin plates.
| |
| - | </p>
| |
| - | <p>
| |
| - | Re-did the synthesis of the minimal DivJ 60 mers (sequencing results suggest the first DivJM assembly resulted in a single base deletion.
| |
| - | </p>
| |
| - | <p>
| |
| - | Transformation of the CA8 and CA9 miniprep plasmid that we sent in for sequencing. Changed the amount of SOC added. This time we used 250 ul.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/18/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Re-did the restriction digest of the vectors and the minimal DivJ with the restriction enzymes NdeI and AciI.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/19/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Re-did the colony PCR of the TipF plates one last time. Same settings as before.
| |
| - | </p>
| |
| - | <p>
| |
| - | Gel 31: From left to right. Ladder, TipF1, TipF2, TipF3, TipF4, TipF5,TipF6, TipF7, TipF8, TipF9, TipF10, Negative Control from TipF plate, Positive
| |
| - | Control.
| |
| - | </p>
| |
| - | <p>
| |
| - | <strong>9/20/13</strong>
| |
| - | </p>
| |
| - | <p>
| |
| - | Colony PCR of the Minimal DivJ Plate:
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix for pVCPFPC-4 plasmids:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 60 ul 2x OneTaq
| |
| - | </p>
| |
| - | <p>
| |
| - | - 24 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul Pvan-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Mastermix for pXYFPC-2 plasmids:
| |
| - | </p>
| |
| - | <p>
| |
| - | - 60 ul 2x OneTaq
| |
| - | </p>
| |
| - | <p>
| |
| - | - 24 ul MilliQ water
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul Pxyl-for primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | - 12 ul eGYC-1 primer at 10 uM concentration
| |
| - | </p>
| |
| - | <p>
| |
| - | Touchdown PCR Protocol:
| |
| - | </p>
| |
| - | <table border="0" cellspacing="0" cellpadding="0" width="624">
| |
| - | <tbody>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Temp
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 60°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 94°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 53°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 68°C
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 4°C
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Time
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:15
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 0:30
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | 5:00
| |
| - | </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>
| |
| - | Hold
| |
| - | </p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | <p>
| |
| - | <strong> </strong>
| |
| - | 15 cycles 15 cycles
| |
| - | </p>
| |
"
