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| − | {{Template_All_Teams}}
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| − | <html>
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| − | </div></div></div></div></div></div></div></div></div></div>
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| − | </html>
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| − | {{Team:UMarylandClasses}}
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| − | {{Team:UMarylandMenu}}
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| − | <html>
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| | | | |
| − | <!--Attention! If you are not part of the website team, you are NOT allowed to touch anything above this line without the express permission of Best Kohai.-->
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| − |
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| − | <style type="text/css">
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| − | h1,h2,h3,h4,p,a {
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| − | text-decoration: none;
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| − | }
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| − | h1 {
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| − | font-size: 250%;
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| − | }
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| − | h2 {
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| − | font-size: 200%;
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| − | }
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| − | h3 {
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| − | font-size: 150%;
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| − | }
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| − | h4 {
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| − | font-size: 125%;
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| − | }
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| − | p {
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| − | font-size: 100%;
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| − | }
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| − | body {
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| − | background-image: -webkit-linear-gradient(bottom, #ffffff 0%, #2BB1FF 100%);
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| − | background-size: 1000% ;
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| − | background-repeat: no-repeat;
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| − | }
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| − |
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| − | #layer1 {
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| − | overflow:hidden;
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| − | position:relative;
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| − | width:100%;
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| − | margin:0px;
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| − | padding:20px;
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| − | background-color: #89e1ff;
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| − | min-height:42px;
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| − | top:-245px;
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| − | border-top:2px solid black;
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| − | }
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| − | #layer2 {
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| − | overflow:hidden;
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| − | position:relative;
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| − | width:100%;
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| − | margin:0px;
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| − | padding:20px;
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| − | background-color:#ffffff;
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| − | min-height:42px;
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| − | top:-245px;
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| − | border-top:2px solid black;
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| − | }
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| − | #layer3 {
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| − | overflow:hidden;
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| − | position:relative;
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| − | width:100%;
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| − | margin:0px;
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| − | padding:20px;
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| − | background-color:#7fcff9;
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| − | min-height:42px;
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| − | top:-245px;
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| − | border-top:2px solid black;
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| − | }
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| − | #layer4 {
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| − | overflow:hidden;
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| − | position:relative;
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| − | width:100%;
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| − | margin:0px;
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| − | padding:20px;
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| − | background-color:#ffffff;
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| − | min-height:42px;
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| − | }
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| − | #contentbox{
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| − | width: 80%;
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| − | max-width: 1900px;
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| − | min-width: 1000px;
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| − | text-align: justify;
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| − | margin:auto;
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| − | font-family: "Palatino Linotype", "Book Antiqua", Palatino, serif;
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| − | }
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| − | #cover {
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| − | overflow:hidden;
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| − | position:relative;
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| − | width:100%;
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| − | margin:auto;
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| − | padding:0px;
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| − | background-image: url("https://static.igem.org/mediawiki/2015/9/97/Resultsq.png");
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| − | background-size: 100% ;
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| − | background-repeat: no-repeat;
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| − | height: 100%;
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| − | width:100%;
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| − | min-height:800px;
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| − | color: #0DFF00;
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| − | font-family: Verdana, Geneva, sans-serif;
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| − | font-size:xx-large;
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| − | top:-200px;
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| − | }
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| − |
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| − | #bar{
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| − | display: inline-block;
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| − | opacity:1;
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| − | background-color: rgba(255,255,255,.5);
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| − | color:black;
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| − | border-top:4px solid black;
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| − | border-bottom:4px solid black;
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| − | width:100%;
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| − | text-align:center;
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| − | height:200px;
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| − | position:absolute;
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| − | top:200px;
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| − | text-shadow: 4px 4px 4px white;
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| − | }
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| − | ul.a {list-style-type: circle;font-size:24px;color:black}
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| − | </style>
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| − |
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| − | <div id='cover'>
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| − | <div id='bar'>
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| − | <p style="font-size:64px"><b>Protocols</b>
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| − | </div>
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| − |
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| − |
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| − | </div>
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| − | </div>
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| − |
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| − |
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| − | <div id='contentbox'>
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| − | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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| − | <b>Miniprep</b></a>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Materials:
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| − | <ul class="a">
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| − | <li>- 250 µL Buffer P1</li>
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| − | <li>- 250 µLBuffer P2</li>
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| − | <li>- 350 µL Buffer N3</li>
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| − | <li>- 750 µL Buffer PE</li>
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| − | <li>- 100 µL DDH2O</li>
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| − | </ul>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Procedure:
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| − | <ul class="a">
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| − | <li>- Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube) by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)</li>
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| − | <li>- Resuspend pellet in 250 µL Buffer P1</li>
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| − | <li>- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear </li>
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| − | <li>- Do not allow reaction to proceed for more than 5 mins</li>
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| − | <li>- If using Lyse Blue, reagent, solution will turn blue</li>
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| − | <li>- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
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| − | </li>
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| − | <li>- Centrifuge for 10 mins at 13000 rpm</li>
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| − | <li>- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting</li>
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| − | <li>- Centrifuge for 60 secs and discard flow through</li>
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| − | <li>- Wash the Q1A prep column with 750 µL Buffer PE</li>
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| − | <li>- Centrifuge for 60 secs and discard flow through</li>
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| − | <li>- Centrifuge for 60 secs to remove residual wash buffer</li>
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| − | <li>- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column</li>
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| − | <li>- Let stand for 1 min and centrifuge for 1 min</li>
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| − | <li>- Repeat steps 12 and 13</li>
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| − | </ul>
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| − | <br>
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| − | <br>
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| − | </div>
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| − |
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| − | <div id='contentbox'>
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| − | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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| − | <b>Ligation</b></a>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Materials:
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| − | <ul class="a">
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| − | <li>- 2 µL PSBIA3 digest</li>
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| − | <li>- 2 µL upstream digest (pBAD/ sRNBC)</li>
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| − | <li>- 2 µLdownstream digest (miraculin / const_GFP)</li>
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| − | <li>- 1 µL T4 DNA ligase</li>
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| − | <li>- 2 µL T4 DNA ligase 10x rxn buffer</li>
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| − | </ul>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Procedure:
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| − | <ul class="a">
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| − | <li>- Combine reagents in a clean PCR tube</li>
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| − | <li>- Let stand 10 mins at room temperature/ no heat kill</li>
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| − |
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| − | </ul>
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| − | <br>
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| − | <br>
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| − | </div>
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| − |
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| − | <div id='contentbox'>
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| − | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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| − | <b>Transformation</b></a>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Materials:
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| − | <ul class="a">
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| − | <li>- 50 µL cells (DH5alpha)</li>
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| − | <li>- 2 µL DNA </li>
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| − | </ul>
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| − | <p style="font-size:24px;text-align:left;text-decoration: underline;">
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| − | Procedure:
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| − | <ul class="a">
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| − | <li>- Add DNA to cells</li>
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| − | <li>- Incubate on ice for 30 minutes</li>
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| − | <li>- Heat shock at 42° fro 30 seconds</li>
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| − | <li>- Add 1 mL SOC media to the cells</li>
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| − | <li>- Incubate for 60 minutes at 37°</li>
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| − | <li>- Plate 200 µL </li>
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| − | <li>- Incubate at 37°</li>
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| − |
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| − | </ul>
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| − | <br>
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| − | <br>
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| − | </div>
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| − | </div>
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| − |
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| − | </html>
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