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| − | {{NCKU_Tainan/header}} {{NCKU_Tainan/navbar}} {{NCKU_Tainan/style}}
| + | r: white !important; |
| − | | + | |
| − | <html>
| + | |
| − | | + | |
| − | <head>
| + | |
| − | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/protocal?action=raw&ctype=text/css">
| + | |
| − | </head>
| + | |
| − | | + | |
| − | <body>
| + | |
| − | | + | |
| − | <!--Page_Content-->
| + | |
| − | <div class="container content">
| + | |
| − | <div class="navbar-example">
| + | |
| − | <div class="row">
| + | |
| − | | + | |
| − | <div class="col-10">
| + | |
| − | <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example">
| + | |
| − | <div class="container">
| + | |
| − | <h1 class="head2">Protocol</h1>
| + | |
| − | <div id="list-item-1">
| + | |
| − | </br>
| + | |
| − | <div class="row">
| + | |
| − | <a class="btn col-md-12" data-toggle="collapse" href="#PCR" role="button"
| + | |
| − | aria-expanded="false" aria-controls="multiCollapseExample1">
| + | |
| − | PCR
| + | |
| − | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
| + | |
| − | </a>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="collapse multi-collapse" id="PCR">
| + | |
| − | <p class="pcontent"></br></br>1. Gently mix the following reaction by pipetting and
| + | |
| − | centrifuge briefly.</p>
| + | |
| − | <table class="card card-body">
| + | |
| − | <tr>
| + | |
| − | <th></th>
| + | |
| − | <th>20 μl system</th>
| + | |
| − | <th>50 μl system</th>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Template</td>
| + | |
| − | <td>12～20 ng</td>
| + | |
| − | <td>30～50 ng</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Forward primer</td>
| + | |
| − | <td>1.0 μl</td>
| + | |
| − | <td>2.5 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Reverse primer</td>
| + | |
| − | <td>1.0 μl</td>
| + | |
| − | <td>2.5 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>dNTP</td>
| + | |
| − | <td>1.6 μl</td>
| + | |
| − | <td>4.0 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>10x Buffer</td>
| + | |
| − | <td>2.0 μl</td>
| + | |
| − | <td>5.0 μl</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p class="pcontent"></br>Program of KOD DNA polymerase</p>
| + | |
| − | <table class="card card-body">
| + | |
| − | <tr>
| + | |
| − | <th>Temperature</th>
| + | |
| − | <th>Time</th>
| + | |
| − | <th></th>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>94 ℃</td>
| + | |
| − | <td>3 min.</td>
| + | |
| − | <td rowspan="3">25～30 cycles</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>94 ℃</br>(Denaturation)</td>
| + | |
| − | <td>40 sec</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>57.5 ℃</br>(Annealing)</td>
| + | |
| − | <td>30 sec</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>72 ℃</br>(Extension)</td>
| + | |
| − | <td>Depend on sequence size</br>(2 kbp/min. for Taq)</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>72 ℃</td>
| + | |
| − | <td>5 min.</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>4 ℃</td>
| + | |
| − | <td>∞</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </table>
| + | |
| − | <p class="pcontent"></br>2. Confirm the size of the digested product by gel
| + | |
| − | electrophoresis.</br></br>
| + | |
| − | 3. Gel purification of the target size.</br></br></p>
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| − | | + | |
| − | </div>
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| − | | + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="list-item-2">
| + | |
| − | | + | |
| − | </br>
| + | |
| − | <div class="row">
| + | |
| − | <a class="btn col-md-12" data-toggle="collapse" href="#Plasmid_Construction" role="button"
| + | |
| − | aria-expanded="false" aria-controls="multiCollapseExample1">
| + | |
| − | Plasmid Construction
| + | |
| − | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
| + | |
| − | </a>
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| − | </div>
| + | |
| − | | + | |
| − | <div class="collapse multi-collapse" id="Plasmid_Construction">
| + | |
| − | </br></br>
| + | |
| − | <p class="pcontent">1. Digestion (vector)</p>
| + | |
| − | <table class="card card-body">
| + | |
| − | <tr>
| + | |
| − | <th>Plasmid</th>
| + | |
| − | <th>200 ng</th>
| + | |
| − | <th>1000 ng</th>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>EcoRI / SpeI</td>
| + | |
| − | <td>0.2 μl</td>
| + | |
| − | <td>1 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>XbaI / PstI</td>
| + | |
| − | <td>0.2 μl</td>
| + | |
| − | <td>1 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>CutSmart Buffer</td>
| + | |
| − | <td>2 μl</td>
| + | |
| − | <td>5 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>ddH2O</td>
| + | |
| − | <td>Up to 20 μl</td>
| + | |
| − | <td>Up to 50 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Digestion at 37℃ for 2.5hr.</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </table>
| + | |
| − | | + | |
| − | </br>
| + | |
| − | <p class="pcontent">2. Digestion (insert)</p>
| + | |
| − | <table class="card card-body">
| + | |
| − | <tr>
| + | |
| − | <th>Plasmid</th>
| + | |
| − | <th>200 ng</th>
| + | |
| − | <th>1000 ng</th>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>EcoRI / XbaI</td>
| + | |
| − | <td>0.2 μl</td>
| + | |
| − | <td>1 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>SpeI / PstI</td>
| + | |
| − | <td>0.2 μl</td>
| + | |
| − | <td>1 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>CutSmart Buffer</td>
| + | |
| − | <td>2 μl</td>
| + | |
| − | <td>5 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>ddH2O</td>
| + | |
| − | <td>Up to 20 μl</td>
| + | |
| − | <td>Up to 50 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Digestion at 37℃ for 2.5hr.</td>
| + | |
| − | <td></td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </table>
| + | |
| − | | + | |
| − | </br>
| + | |
| − | <p class="pcontent">3.Confirm the size of the digested product by gel
| + | |
| − | electrophoresis.</br></br>
| + | |
| − | 4. Gel purification of the target size.</br></br>
| + | |
| − | 5. Ligation</br>
| + | |
| − | | + | |
| − | <table class="card card-body">
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Vector (2 kbp)</td>
| + | |
| − | <td rowspan="2">molar ratio = 1:3</br>(can up to 1:10 depending on the
| + | |
| − | DNA sizes)</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Insert (1.5 kbp)</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Quick Ligase Reaction Buffer (2X)*</td>
| + | |
| − | <td>10 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>Quick Ligase</td>
| + | |
| − | <td>1 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td>ddH2O</td>
| + | |
| − | <td>Up to 20 μl</td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | </table>
| + | |
| − | | + | |
| − | </br>
| + | |
| − | <p class="pcontent">6. Transform the product by heat shock.</br>
| + | |
| − | | + | |
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| − | </div>
| + | |
| − | </div>
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| − | | + | |
| − | | + | |
| − | <div id="list-item-3">
| + | |
| − | </br>
| + | |
| − | <div class="row">
| + | |
| − | <a class="btn col-md-12" data-toggle="collapse" href="#Extraction" role="button"
| + | |
| − | aria-expanded="false" aria-controls="multiCollapseExample1">
| + | |
| − | PCR Clean-Up & Gel Extraction
| + | |
| − | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
| + | |
| − | </a>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="collapse multi-collapse" id="Extraction">
| + | |
| − | | + | |
| − | </br></br><h3>Gel Dissociation</h3></br>
| + | |
| − | <p class="pcontent">1. Gel Extraction</br></br>
| + | |
| − | a. Excised the DNA fragment from the agarose gel.</br></br>
| + | |
| − | b. Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube.</br></br>
| + | |
| − | c. Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.
| + | |
| − | Incubate
| + | |
| − | at 55~60℃ for 10 minutes (or until the gel slice has completely dissolved).</br></br>
| + | |
| − | d. During the incubation, mixed by vortexing the tube every 2~3 minutes.</br></br>
| + | |
| − | e. Cooled the dissolved sample mixture to the room temperature.</br></br>
| + | |
| − | </p>
| + | |
| − | <h3>DNA Binding</h3></br>
| + | |
| − | <p class="pcontent">2. Placed a PG Column in a Collection Tube. Apply the
| + | |
| − | supernatant to the PG
| + | |
| − | Column
| + | |
| − | by decanting or pipetting.</br></br>
| + | |
| − | 3. Centrifuged at 16,000 xg for 30 seconds.</br></br>
| + | |
| − | 4. Discarded the flow-through and place the PG Column back into the same
| + | |
| − | collection
| + | |
| − | tube.</br></br></p>
| + | |
| − | <h3>Wash</h3></br>
| + | |
| − | <p class="pcontent">6. Added 400 μl of the Buffer W1 into the PG Column.</br></br>
| + | |
| − | 7. Centrifuged at 16,000 xg for 30 seconds.</br></br>
| + | |
| − | 8. Discarded the flow-through and place the PG Column back into the same
| + | |
| − | collection
| + | |
| − | tube.</br></br>
| + | |
| − | 9. Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.</br></br>
| + | |
| − | 10. Centrifuged at 16,000 xg for 30 seconds.</br></br>
| + | |
| − | 11. Discarded the flow-through and place the PG Column back into the same
| + | |
| − | collection tube.</br></br>
| + | |
| − | 12. Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer
| + | |
| − | W2.</br></br></p>
| + | |
| − | <h3>Elution</h3></br>
| + | |
| − | <p class="pcontent">13. To elute the DNA, placed the PG Column in a clean 1.5 ml
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| − | microcentrifuge
| + | |
| − | tube.</br></br>
| + | |
| − | 14. Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of each PG
| + | |
| − | Column, let it stand for at least 2 minutes, and centrifuge at 16,000 xg for 2
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| − | min.</br></br></p>
| + | |
| − | </p>
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| − | | + | |
| − | </div>
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| − | </div>
| + | |
| − | </br>
| + | |
| − | <div class="row">
| + | |
| − | <a class="btn col-md-12" data-toggle="collapse" href="#Preparation" role="button"
| + | |
| − | aria-expanded="false" aria-controls="multiCollapseExample1">
| + | |
| − | Competent Cell Preparation
| + | |
| − | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
| + | |
| − | </a>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="collapse multi-collapse" id="Preparation">
| + | |
| − | <p class="pcontent"></br></br>･ For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell</br></br>
| + | |
| − | 1. Streak out wild type E. coli on a plate (LB plate without antibiotics) overnight and pick one colony into 3 ml of media (LB) and grow overnight.</br></br>
| + | |
| − | 2. Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at 37 ℃.</br></br>
| + | |
| − | 3. When the OD600 nm up to 0.35, put the cells on ice immediately.</br></br>
| + | |
| − | 4. Spin the cells at 4℃for 10 minutes at 4000 rpm.</br></br>
| + | |
| − | 5. Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer 1(TFB1)</br></br>
| + | |
| − | 6. Leave nicely suspended bugs on ice for 10 minutes.</br></br>
| + | |
| − | 7. Spin the cells at 4℃ for 10 min. at 4000 rpm.</br></br>
| + | |
| − | 8. Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).</br></br>
| + | |
| − | 9. Leave on immediately on ice for 30 minutes.</br></br>
| + | |
| − | 10. Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.</br></br>
| + | |
| − | 11. Store the frozen cells in the -80℃ freezer.</br></br></p>
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| − | </div>
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| − | </div>
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| − | <style>
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| − | .head2 {
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| − | color: white;
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| − | width: 26%;
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| − | font-size: 50px;
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| − | /* font-weight: 700; */
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| − | border-bottom: 10px solid #7ae26f !important;
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| − | position: relative;
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| − | }
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| − | /* paragraph content*/
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| − | h10 {
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| − | color: white;
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| − | line-height: 150%;
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| − | font-size: 35px;
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| − | .navbar {
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| − | padding-top: 10px;
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| − | margin-bottom: 0;
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| − | .navbar-brand {
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| − | font-size: 30px;
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| − | .nav-link {
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| − | font-size: 20px;
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| − | a.dropdown-item {
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| − | color: #4F7F52;
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| − | a.dropdown-item:active {
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| − | background-color: #4F7F52;
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| − | .caret {
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| − | display: inline-block;
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| − | margin-left: 2px;
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| − | vertical-align: middle;
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| − | border-top: 4px solid;
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| − | border-right: 4px solid transparent;
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| − | border-left: 4px solid transparent;
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| − | }
| + | |
| − | | + | |
| − | /*滑到navbar就展開*/
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| − | .dropdown-menu li:hover .sub-menu {
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| − | visibility: visible;
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| − | }
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| − | .dropdown:hover .dropdown-menu {
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| − | display: block;
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| − | }
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| − | .head {
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| − | color: white;
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| − | width: 50%;
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| − | font-size: 50px;
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| − | font-weight: 700;
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| − | border-bottom: 10px solid #7ae26f;
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| − | position: relative;
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| − | margin-top: 100px;
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| − | }
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| − | .container.content {
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| − | margin-top: 80px;
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| − | }
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| − | @media (min-width: 992px) {
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| − | .navbar {
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| − | padding-left: 80px;
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| − | padding-right: 80px;
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| − | }
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| − | }
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| − | @media (max-width: 768px) {
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| − | .navbar-right form {
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| − | display: none;
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| − | }
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| − | }
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| − | @media (max-width: 568px) {
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| − | footer {
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| − | text-align: center;
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| − | }
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| − | .list-group {
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| − | display: none;
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| − | }
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| − | }
| + | |
| − | | + | |
| − | /*folded-corner*/
| + | |
| − | .post {
| + | |
| − | position: relative;
| + | |
| − | }
| + | |
| − | | + | |
| − | .folded-corner {
| + | |
| − | position: absolute;
| + | |
| − | bottom: 0px;
| + | |
| − | right: 0px;
| + | |
| − | border-width: 0;
| + | |
| − | border-style: solid;
| + | |
| − | background: hsla(260, 100%, 100%, 0.2);
| + | |
| − | box-shadow: 0px -4px 0px rgba(0, 0, 0, 0.3), -1px -4px 0px rgba(0, 0, 0, 0.1);
| + | |
| − | border-radius: 15px 0 0 0;
| + | |
| − | border-color: transparent #B9DEBB transparent transparent;
| + | |
| − | transition: border-width 0.2s ease-out;
| + | |
| − | }
| + | |
| − | | + | |
| − | .post:hover .folded-corner {
| + | |
| − | border-width: 40px 40px 0 0;
| + | |
| − | }
| + | |
| − | | + | |
| − | .photo .folded-corner {
| + | |
| − | background: hsla(260, 5%, 75%, 0.5);
| + | |
| − | }
| + | |
| − | | + | |
| − | [class*="col-"] {
| + | |
| − | float: left;
| + | |
| − | padding: 13px;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.list-group-item:visited {
| + | |
| − | color: white;
| + | |
| − | }
| + | |
| − | | + | |
| − | a:hover {
| + | |
| − | background-color: transparent;
| + | |
| − | }
| + | |
| − | | + | |
| − | .list-group {
| + | |
| − | margin-top: 100px;
| + | |
| − | }
| + | |
| − | | + | |
| − | .col-2.side {
| + | |
| − | padding: 0;
| + | |
| − | }
| + | |
| − | | + | |
| − | .list-group-item {
| + | |
| − | padding: .55rem .35rem;
| + | |
| − | background-color: #272625;
| + | |
| − | color: white;
| + | |
| − | border: none;
| + | |
| − | font-size: 19px;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.list-group-item.list-group-item-action {
| + | |
| − | background-color: transparent;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.list-group-item.list-group-item-action:hover {
| + | |
| − | color: #98CC9B;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.list-group-item.list-group-item-action.active {
| + | |
| − | background-color: transparent;
| + | |
| − | color: #7ae26f;
| + | |
| − | }
| + | |
| − | | + | |
| − | .scrollspy-example h4 {
| + | |
| − | font-size: 2rem;
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | img.contentimg {
| + | |
| − | width: 100%;
| + | |
| − | margin: 20px 0;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.link {
| + | |
| − | font-size: 30px;
| + | |
| − | color: #4F7F52;
| + | |
| − | }
| + | |
| − | | + | |
| − | #list-item-1 {
| + | |
| − | padding-top: 0px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #list-item-2 {
| + | |
| − | padding-top: 0px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #list-item-3 {
| + | |
| − | padding-top: 0px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #list-item-4 {
| + | |
| − | padding-top: 0px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #list-item-5 {
| + | |
| − | padding-top: 0px;
| + | |
| − | }
| + | |
| − | | + | |
| − | a.reference {
| + | |
| − | color: #4F7F52;
| + | |
| − | }
| + | |
| − | | + | |
| − | .btn {
| + | |
| − | background-color: #98CC9B;
| + | |
| − | color: #272625;
| + | |
| − | border: none;
| + | |
| − | font-size: 1.5rem;
| + | |
| − | }
| + | |
| − | | + | |
| − | .btn:hover {
| + | |
| − | color: white;
| + | |
| − | background-color: transparent;
| + | |
| − | border-color: transparent;
| + | |
| − | border-bottom: 3px;
| + | |
| − | border-bottom-color: #98CC9B;
| + | |
| − | border-bottom-style: solid;
| + | |
| − | border-radius: 0rem;
| + | |
| − | }
| + | |
| − | | + | |
| − | .btn.focus,
| + | |
| − | .btn:focus {
| + | |
| − | box-shadow: none;
| + | |
| − | }
| + | |
| − | | + | |
| − | td {
| + | |
| − | text-align: center;
| + | |
| − | color: black !important;
| + | |
| − | border: 2px solid black !important;
| + | |
| − | background-color: white !important;
| + | |
| | } | | } |
| | | | |
