User:Neil J. Parikh/Lab Notebook

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 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style='margin-right:26.1pt'>Pipetting &amp; Making
 Solutions</p>
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 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
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 <p class=ExperimentNumber>Pg. 6</p>
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<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; font-weight:normal;font-style:normal'>Pipetting</span></u></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Learned to use pipettes properly</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Make sure to wipe down pipettes between solutions (with Ethanol)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Change tips every time you go between solutions</span></span></p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; font-weight:normal;font-style:normal'>Solutions</span></u></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Task: Make 0.3M KCl</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>i.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Salt (KCl): CAS 7447-40-7</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>ii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>MW: 74.56g/mol</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>iii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>2.2368 g / 100mL</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

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 <p class=MsoTitle style='margin-right:26.1pt'>Plate Creation &amp; Cell
 Culture</p>
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 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
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 <p class=ExperimentNumber>Pg. 7</p>
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<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; font-weight:normal;font-style:normal'>Steps for creating a plate</span></u></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>:</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Dissolve 25g LB in 800mL of H<sub>2</sub>O</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Bring the final volume to 1L in a 2L flask</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Add 15g Bactoagar, cover with foil, autoclave for 20-30 minutes</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Bring back to lab, cool to 50</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>C </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>while stirring</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add100mg AMP &amp; let swirl until dissolved</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Pour it into the plates (about 2-3mm deep)</span></span></p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; font-weight:normal;font-style:normal'>Cell Culture</span></u></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>:</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Use solution of LB with AMP</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Ensure to keep the bags sealed to keep everything sterile </span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>*Note once a bag of plates has been opened, the whole thing must be used</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>The tubes allow the solution to be aerated</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>The tubes with bacteria should look cloudy upon visual inspection—can be confirmed with cell density measurement (using the Spec)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Sterilize the autopipette, set to 1mL (make a 2mL culture, however)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Add 2mL of the LB (for a miniprep); you will have to add 1mL, two times</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Label one as the control and another as your experimental sample</span></span></p>

<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>*Label the tubes with your Initials, Sample Name (control or experimental) and the date</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Dip the culture scraper in ethanol and flame lightly (2x) to sterilize</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>e.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Let the scraper cool for a few seconds</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>f.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Scrape off a tiny bit of bacteria off of the plate and place the scraper into the tube and shake around to ensure the bacteria breaks off into the tube</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>g.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Set incubator at 37</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>C overnight and let shake gently for 12-14hrs</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

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 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style='margin-right:26.1pt'>Transformation of E.coli with
 Recombinant DNA</p>
 </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber>Pg. 9</p>
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<p class=MsoNormal style='margin-left:.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>*note, </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>the heat shock</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> to the tubes is very important for transcription to occur</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> <u>+ Controls</u>:</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_I13522 : pTetGFP</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_J45220</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> <u>Group 1:</u></span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_R0011</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_J45219</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> <u>Group 2:</u></span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_I14032 / BBa_R0051 (same)</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_J06702</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> <u>Group 3:</u></span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_J04500</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> BBa_J04630 / BBa_I13401</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Rima &amp; I did the transformation for Group 2:</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_I14032</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name: </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>promoter P(Lac) IQ</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Sequence: </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>promoter</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'> </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>(-35, -10 bases before coding)</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>: 2007, Plate 2, Well 17K</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid: </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>pSB2K3</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_R0051</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name:</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> promoter (lamda cl regulated)</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Sequence:</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> R0051 promoter</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location: </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>2007, Plate 1, Well 9C</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><i>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></i><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid:</span></span><i> </i>pSB1A2</p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>III.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>BBa_J06702</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name:</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> mCherry, bacterial with RBS &amp; forward terminator</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Sequence</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>: </span></span></p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:159.5pt;border-collapse:collapse;border:none'>
<tr>
 <td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal'>RBS</span></span></p>
 </td>
 <td width=85 valign=top style='width:64.05pt;border:solid windowtext 1.0pt;
 border-left:none;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal'>mCherry</span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt;
 border-left:none;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal'>Terminator</span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt;
 border-left:none;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal'>Terminator</span></span></p>
 </td>
</tr>
<tr>
 <td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt;
 border-top:none;padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0034</span></span></p>
 </td>
 <td width=85 valign=top style='width:64.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>J06504</span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0010</span></span></p>
 </td>
 <td width=101 valign=top style='width:76.05pt;border-top:none;border-left:
 none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext;
 font-weight:normal;font-style:normal'>B0012</span></span></p>
 </td>
</tr>

</table>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location:</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> 2007, Plate 2, Well 11D</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>d.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid:</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> pSB1A2</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>IV.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Cells Used: E.coli: JM109 (Promega); Catalog #: L2001</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>V.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>3 Plates Made:</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Control: 100&#956;L LB / Cell</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Test Plate 1: 100&#956;L LB/ Cell</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Test Plate 2: 1&#956;L cell : 99&#956; H<sub>2</sub>O</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Actual Transformation Procedure:</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>If using DNA from the registry, dilute with 9&#956;L of H<sub>2</sub>O. If using our own DNA, use 200ng. </span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>inject the water directly into the well, titerate, and then pipette it out</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Thaw 100&#956;L competent cells on ice. (50</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&#956;L with our own/ 100</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956; if commercial cells used)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>III.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Mix competent cells with 50</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956; CaCl<sub>2</sub> (only if using our own cells)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>IV.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 4</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L of registry DNA, or 200ng of our own DNA to the cells. </span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>V.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Put in the ice bath for about 20 minutes. </span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VI.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Remove and immediately put in the hot water bath (42</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>C) to heat shock for 45-60 sec.</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VII.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Place back in the ice bath for 2 minutes. </span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VIII.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 900 &#956;L ice cold, antibiotic free LB to the cells</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>IX.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate at 37</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>C for 90 minutes in the shaking incubator.</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>X.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Dilute as desired, make the necessary controls, and plate cells using 100 &#956;L / plate. (200 if not commercial)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>XI.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate upside down (so that the label you wrote on it is face up) at 37</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>C for 18-24 hours, then refrigerate. </span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>The controls should not have bacteria growing on them!</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse;
border:none'>
<tr>
 <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style='margin-right:26.1pt'>Miniprep</p>
 </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber>Pg. 12</p>
 </td>
</tr>

</table>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>*Start with Parts: E0241 (x3), R0010, R0040, C0040 from the registry*</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>E0241: PoPs to GFP Converter</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>RBS: B0032</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>GFP: E0040</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Terminator: B0016</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>R0010: Promoter Lac I (Regulator) </span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>R0040: Promoter (tetR, negative)</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>C0040: Tetracycline repressor from transposon Tn10 (+LVA) [note that +LVA is a degradation tag]</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Look out for:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'> </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>Control should not be cloudy at all, culture should have gas bubbles, should be cloudy, especially at the bottom</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> *If you start seeing sediment at the bottom, the cells </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>may</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> have lysed and let proteins into the tube*</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> </span></span></p>

<p class=MsoNormal style='text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Procedure (General):</span></span></p>

<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Use the QIAGEN Kit to separate the plasmid DNA (so it should not have much chromosomal DNA)</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Grow cells for 12 to 14 hours</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Suspend cells in </span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>i.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 1 (with glucose &amp; EDTA)</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>ii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 2 (SDS-detergent, NaOH) [SDS gets rid of the membrane and NaOH denatures the plasmid and DNA]</span></span></p>

<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>iii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>buffer 3 (K Acetate / Acetic Acid)</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-things become an insoluble precipitate</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-renature the DNA/plasmid</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-only the plasmid DNA will renature properly</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>-the plasmid DNA remains soluble &amp; the chromosomal DNA will become the precipitate</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>III.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Columns: use EtOH to extract DNA</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Procedure (Specific):</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> *make sure to check your buffers for salt precipitation—this may indicate a change in concentration*</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Get solution and put 1.5mL into an Eppendorf tube to centrifuge </span></span></p>

<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>-after centrifuging for 30 seconds @ 13k RPM, remove the supernatant</span></span></p>

<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>-add more solution, and centrifuge again using same procedure</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 250</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L PI to tube after the supernatant is </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>fully removed</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>III.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 250 &#956;L P2, and invert 4 to 6 times</span></span></p>

<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>-immediately add 350</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&#956;L N3, and invert again 4 to 6 times</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>IV.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Dispose of the flow through, add 0.5mL of buffer PB (that is </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>milliliter not microliter!)</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'> then centrifuge for 30-60 seconds</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>V.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 750</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L PE, then centrifuge again, remove the waste, centrifuge again</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VI.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add 50</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>&#956;L BE buffer</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VII.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Measure the DNA concentration using Nanodrop</span></span></p>

<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VIII.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Ethanol Precipitation </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>(see separate procedure)</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal'>&nbsp;</span></span></p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse;
border:none'>
<tr>
 <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=MsoTitle style='margin-right:26.1pt'>Ethanol Precipitation (for
 &gt;200bp)</p>
 </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
 padding:0in 5.4pt 0in 5.4pt'>
 <p class=ExperimentNumber>Pg. 15</p>
 </td>
</tr>

</table>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> *used to purify the DNA (really small fragments) or to increase the concentration of it</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>I.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Add:</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>i.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>3 volumes of COLD 100% EtOH</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>ii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1/10 volume of 3M sodium acetate</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>iii.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1 &#956;L GlycoBlue</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>II.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Incubate for 1 hr @ 80</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>C</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>III.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Centrifuge for 30min @ 0</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>C</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>IV.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Remove supernatant</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>V.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Air Dry</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>VI.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Resuspend in H<sub>2</sub>O</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Specific Procedure Example:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> For 50 &#956;L:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> Add

   150 &#956;L EtOH</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 20 &#956;L NaAc.</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> For 100 &#956;L:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> Add 300 &#956;L EtOH</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 40 &#956;L NaAc.</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0

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 <p class=MsoTitle style='margin-right:26.1pt'>Gel Electrophoresis</p>
 </td>
 <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt;
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 <p class=ExperimentNumber>Pg. 16</p>
 </td>
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</table>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> *applying an electric field over the gel will separate fragments by size</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> *compare the movement of the DNA to the ladder</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'> </span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext;font-weight:normal'>*ensure that the well is on the negative side!*</span></u></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>DNA has a (-) charge because of the phosphate groups</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>moves toward the anode (+) and away from the cathode (-)</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>DNA is separated by size, distance ~ 1 / molecular weight</span></span></p>

<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>100bp-50kbp</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>Gel acts as a sieve: 0.3% large fragments to 2% small fragments</span></span></p>

<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>after the gel is made, put it in the microwave for 1minute, then </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>cool the bottle in H­<sub>2</sub>O, and take the lid off</span></span></p>

<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Casting Gel:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 1% Agarose, 0.5g Agarose</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 50mL TBE + SYBR Safe</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Dilute the 5xTBE to 0.5x</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>Add 2 &#956;L of the loading dye!</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'>&nbsp;</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Ladder Formula:</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'> </span></span><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; font-style:normal'>1 &#956;L 1kbase ladder</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 2 &#956;L BlueJuice Dye</span></span></p>

<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal;font-style:normal'> 7 &#956;L H<sub>2</sub>O</span></span></p>

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