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                <article id = "miniprep">
                    <h3>Miniprep</h3>
                    <ol>
                    <li>Resuspend pelleted bacterial cells in 250 µl Buffer PI and transfer to a microcentrifuge tube.</li>
                    <li>Add 250 µl Buffer P2 and gently invert tube 4-6 times to mix</li>
                    <li>Add 350 µl Buffer N3 and invert immediately 4-6 times</li>
                    <li>Centrifuge for 10 min at 13,000 rpm</li>
                    <li>Add supernatant to QIAprep spin column</li>
                    <li>Centrifuge for 30-60s - Discard flow through</li>
                    <li>Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s - Discard flow through</li>
                    <li>Wash column by adding 0.75 ml Buffer PE and centrifuginh for 30-60s</li>
                    <li>Discard flow through and centrifuge for an additional 1 min to remove residual wash buffer</li>
                    <li>Place column in clean 1.5 ml microcentrifuge tube. To elute DNA add 50 µl water or buffer EB, let stand for 1 min and centrifuge for 1 min.</li>
                    </ol>
                </article>
                <article id = "pcr-pure">              
                    <h3>PCR Purification</h3>

                    <ol>
                    <li>5 volumes of Buffer PB (with ethanol) to 1 volume PCR sample</li>
                    <li>Apply sample to column and spin for 1 min</li>
                    <li>Discard flow through </li>
                    <li>To wash add 0.75 ml of Buffer PE to column and centrifuge for 1 min</li>
                    <li>Discard flow through and place column back in the same tube</li>
                    <li>Cetrifuge for an addtitional minute (Dry Spin)</li>
                    <li>Place column in clean 1.5 ml tube</li>
                    <li>[Elute DNA] Add 30µl water to column, let stand for 1 min and centrifuge for 1 min </li>
                    </ol>    
                </article>
                <article id = "g-extract">
                    <h3>Gel Extraction</h3>
                    <p>QlAquick Gel Extraction Kit </p>
                    <ol>
                    <li>Cut Gel</li>
                    <li>Weigh it in a colorless tube</li>
                    <li>Add 3 volumes Buffer Q G to 1 volime Gel (100mg ~ 100µl)</li>
                    <li>Incubate @ 50° C for 10 min or until completely dissolved (vortex every 2-3 min to help dissolve)</li>
                    <li>Add 1 gel volume isopropanal to the sample and mix</li>
                    <li>Place a QlAquick soin column in a provided 2ml collection tube</li>
                    <li>Place sample in column &amp; spin for 1 min --&gt; discard flow through</li>
                    <li>To wash add 0.75 ml Buffer PE to column &amp; centrifue for 1 min, then dry spin</li>
                    <li>Place column in 1.5 ml tube</li>
                    <li>Add 35µl H2O &amp; centrifuge for 1 min.</li>
                    </ol>
                </article>
                <article id = "e-transform">                    
                    <h3>E. Coli Transformation (Digestion/Ligation)</h3>

                    <pre><code> 
- 10 µl ligation
- 50 µl competent cells
--&gt; 30m on ice
--&gt; 45s heatshock 42° C
--&gt; 2m on ice
- 250 µl of SOC media
--&gt; 1h shake 37° C
                    </code></pre>    
                </article>
                <article id = "y-transform">
                    
<h3>Yeast Transformation</h3>

<table><thead>
<tr>
<th>Reagents</th>
</tr>
</thead><tbody>
<tr>
<td>YPD</td>
</tr>
<tr>
<td>1 M LiOAc</td>
</tr>
<tr>
<td>10X TE pH 7.5</td>
</tr>
<tr>
<td>1X TE pH 7.5, 0.1 M LiOAc</td>
</tr>
<tr>
<td>50% PEG 3350</td>
</tr>
<tr>
<td>DMSO</td>
</tr>
<tr>
<td>Salmon Sperm DNA (ssDNA)</td>
</tr>
</tbody></table>

<ul>
<li>PEG = viscous, pipette slow</li>
<li>boil ssDNA aliquots </li>
</ul>

<p>Previous Day : Grow yeast strain to be transformed in 5-10 mL YPD overnight at 30° C</p>

<ol>
<li>Set up digest to linearize DNA </li>
<li>Dilute O/N culture ~ 1:20 in YPD grow 2-4 hours at 30° C</li>
<li>Prepare ssDNA - boil for 10m cool on ice for at least 10m (10 µl of 10 mg/mL stock per transformation)</li>
<li>Harvest cells in centrifuge - 3000 rpm, 2-5 min</li>
<li>Wash with 1 ml 0.1 M LiOAin TE</li>
<li>Pellet cells - 3000 rpm , 2-5 min</li>
<li>Resuspend pellet in 100 µl 0.1 M LiOAc in TE per 2.5 ml culture, split into 100 µl per epindorph tube for each transformation</li>
<li>to 100 µl cells add 100 µg ssDNA (10 µl of 10 mg/mL sock), 1-5 µl target DNA</li>
<li>Add (in order): 480 µl 50% PEG 3350, 60 µl 10X TE, 60 µl 1 M LiOAc (for final 40% PEG, 1X TE, 0.1 M LiOAc)  <em>Optional</em>: Add 75 µl DMSO <em>definitely did</em></li>
<li>Vortex</li>
<li>Incubate 42° C for 30m &amp; begin drying plates</li>
<li>Pellet (6000 rpm - 2m), discard supernatant (remove PEG completely by pipetting), resuspend in 500 µl YPD (or selective media)</li>
<li>Pellet, discard supernatant, resuspend in risidual ~ 50 µl YPD</li>
<li>Plate on selective media</li>
<li>Incubate 1-3 days</li>
</ol>    
                </article>
                <article id = "e-pcr">
                    <h3>Colony PCR for screening E. Coli</h3>

<p>Pick single colonies ( 5 or so from each plate ) mix in 25 µl H2O in a tube. Use 5 µl in PCR reaction</p>

<table><thead>
<tr>
<th>Reagents</th>
<th>1X</th>
<th>6X</th>
</tr>
</thead><tbody>
<tr>
<td>2X GoTaq Green PCR Master Mix</td>
<td>10 µl</td>
<td>60 µl</td>
</tr>
<tr>
<td>10 µM Forward primer</td>
<td>1 µl</td>
<td>6 µl</td>
</tr>
<tr>
<td>10 µM Reverse primer</td>
<td>1 µl</td>
<td>6 µl</td>
</tr>
<tr>
<td>Water</td>
<td>3 µl</td>
<td>18 µl</td>
</tr>
<tr>
<td>Bacterial cells (template)</td>
<td>5 µl</td>
<td>-----</td>
</tr>
</tbody></table>

<p>Cycle (Varies): </p>

<pre><code>95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever
</code></pre>

<p>load 5 µl onto gel</p>

<p>for all positive bands - take the rest of the bands and inoculate them into an overnight LB (+antibiotic) for miniprep</p>
                    </article>
                <article id = "y-pcr">
                   <h3>Colony PCR for Yeast &amp; Patching</h3>

                    <ol>
                    <li>Number colonies</li>
                    <li>Patch on plate</li>
                    <li>Mix in 10 µl NaOH</li>
                    <li>Boil for 20m</li>
                    <li>PCR</li>
                    </ol>

                    <p>Cycle (varies): </p>
                    <pre><code>
95° C | 5m
30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever
                    </code></pre>
                </article>
                <article id = "g-stocks">
                    <h3>Frozen Glycerol Stocks Yeast</h3>

                    <ol>
                    <li>Grow Overnight in YPD (2-5 mL) then dilute 1:20 in YPD, grow to OD 0.4-0.5</li>
                    <li> Add 350 µl cells to 350 µl sterile 60% glycerol in cryovial, vortex to mix, snap freeze in liquid nitrogen and store at -80° C (but actually we just stuck it in the freezer because nobody will let us use liquid nitrogen).</li>
                    </ol>    
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    erase(logo_rectangle, logo_rectangle.getTotalLength()); 
    erase(logo_line, logo_line.getTotalLength()); 
    erase(logo_promoter, logo_promoter.getTotalLength()); 
    erase(logo_box, logo_box.getTotalLength()); 
    
    draw(logo_rectangle, (logo_rectangle.getTotalLength()), 0,frames); 
    draw(logo_line, logo_line.getTotalLength(), 0, frames); 
    draw(logo_promoter, logo_promoter.getTotalLength(), 0, frames/2); 
    draw(logo_box, logo_box.getTotalLength(), frames/3,frames); 
    for(i = 0; i<17; i++){
        draw(gene_line[i], gene_line[i].getTotalLength(),(frames/3)+i*((2/3)*frames)/17,(8/9)*frames+i*((1/9)*frames/17));
    }
}

//Main Document
//--------------
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