|
|
| Line 6: |
Line 6: |
| | <link href="https://2021.igem.org/Template:Qdai/css/styleCSS?action=raw&ctype=text/css" rel="stylesheet"/> | | <link href="https://2021.igem.org/Template:Qdai/css/styleCSS?action=raw&ctype=text/css" rel="stylesheet"/> |
| | <link href="https://2021.igem.org/Template:Qdai/css/bootstrap-minCSS?action=raw&ctype=text/css" rel="stylesheet"/> | | <link href="https://2021.igem.org/Template:Qdai/css/bootstrap-minCSS?action=raw&ctype=text/css" rel="stylesheet"/> |
| − | <link href="https://2021.igem.org/Template:Qdai/css/g-fontsCSS?action=raw&ctype=text/css" rel="stylesheet"/> | + | <link href="https://2021.igem.org/Template:Qdai/css/g-fontsCSS?action=raw&ctype=text/css" rel="stylesheet"/> |
| | | | |
| | | | |
| | <style> | | <style> |
| − | .main > p > img{
| + | .main>img { |
| − | height: 15vh; | + | height: 25vh; |
| − | } | + | max-width: 100%; |
| | + | } |
| | + | |
| | #HQ_page p { | | #HQ_page p { |
| − | font-family: "Arial",Helvetica,sans-serif;
| + | font-family: "Arial", Helvetica, sans-serif; |
| − | font-size: 16px;
| + | font-size: 16px; |
| − | text-align: justify;
| + | text-align: justify; |
| − | } | + | margin: 12px 0 12px 0; |
| | + | } |
| | + | |
| | .tag { | | .tag { |
| | font-family: 'Gemunu Libre', sans-serif; | | font-family: 'Gemunu Libre', sans-serif; |
| Line 48: |
Line 52: |
| | margin-bottom: 12px; | | margin-bottom: 12px; |
| | } | | } |
| − | #globalWrapper{ | + | |
| | + | #globalWrapper { |
| | font-size: 100%; | | font-size: 100%; |
| | padding-bottom: 0px; | | padding-bottom: 0px; |
| | } | | } |
| − |
| + | |
| − | #mw-content-text>p{ | + | #mw-content-text>p { |
| | display: none; | | display: none; |
| | | | |
| | } | | } |
| − |
| + | |
| − | .mem-link:link, .mem-link:visited, .mem-link:hover, .mem-link:active {
| + | .mem-link:link, |
| − | text-decoration:none;
| + | .mem-link:visited, |
| − | color:#212529;
| + | .mem-link:hover, |
| − | } | + | .mem-link:active { |
| − | .bg{ | + | text-decoration: none; |
| | + | color: #212529; |
| | + | } |
| | + | |
| | + | .bg { |
| | background: url("https://static.igem.org/mediawiki/2021/c/c8/T--Qdai--img--member-top.jpg"); | | background: url("https://static.igem.org/mediawiki/2021/c/c8/T--Qdai--img--member-top.jpg"); |
| | height: 40vh; | | height: 40vh; |
| | } | | } |
| | + | |
| | .CircleClip { | | .CircleClip { |
| − | width:15vw;
| + | width: 15vw; |
| − | height:15vw;
| + | height: 15vw; |
| − | } | + | } |
| − | .s-item{
| + | |
| − | margin-right:2vw;
| + | .s-item { |
| − | }
| + | margin-right: 2vw; |
| | + | } |
| | | | |
| | | | |
| Line 103: |
Line 114: |
| | #bodyContent h2, | | #bodyContent h2, |
| | #bodyContent h3, | | #bodyContent h3, |
| − | #bodyContent h4, | + | #bodyContent p, |
| | #bodyContent h5 { | | #bodyContent h5 { |
| | margin-bottom: 0px; | | margin-bottom: 0px; |
| Line 116: |
Line 127: |
| | /* https://work.smarchal.com/twbscolor/ */ | | /* https://work.smarchal.com/twbscolor/ */ |
| | .navbar { | | .navbar { |
| − | background-color: #98197f00;
| + | background-color: #98197f00; |
| − | }
| + | } |
| − | .navbar .navbar-brand {
| + | |
| − | color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-brand:hover,
| + | |
| − | .navbar .navbar-brand:focus {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar .navbar-text {
| + | |
| − | color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-text a {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar .navbar-text a:hover,
| + | |
| − | .navbar .navbar-text a:focus {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .nav-link {
| + | |
| − | color: #edf2f4;
| + | |
| − | border-radius: .25rem;
| + | |
| − | margin: 0 0.25em;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .nav-link:not(.disabled):hover,
| + | |
| − | .navbar .navbar-nav .nav-link:not(.disabled):focus {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .dropdown-menu {
| + | |
| − | background-color: #151616b6;
| + | |
| − | border-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .dropdown-menu .dropdown-item {
| + | |
| − | color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .dropdown-menu .dropdown-item:hover,
| + | |
| − | .navbar .navbar-nav .dropdown-menu .dropdown-item:focus,
| + | |
| − | .navbar .navbar-nav .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8;
| + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .dropdown-menu .dropdown-divider {
| + | |
| − | border-top-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-nav .nav-item.active .nav-link,
| + | |
| − | .navbar .navbar-nav .nav-item.active .nav-link:hover,
| + | |
| − | .navbar .navbar-nav .nav-item.active .nav-link:focus,
| + | |
| − | .navbar .navbar-nav .nav-item.show .nav-link,
| + | |
| − | .navbar .navbar-nav .nav-item.show .nav-link:hover,
| + | |
| − | .navbar .navbar-nav .nav-item.show .nav-link:focus {
| + | |
| − | color: #a697b8;
| + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-toggler {
| + | |
| − | border-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-toggler:hover,
| + | |
| − | .navbar .navbar-toggler:focus {
| + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | .navbar .navbar-toggler .navbar-toggler-icon {
| + | |
| − | color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-collapse,
| + | |
| − | .navbar .navbar-form {
| + | |
| − | border-color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-link {
| + | |
| − | color: #edf2f4;
| + | |
| − | }
| + | |
| − | .navbar .navbar-link:hover {
| + | |
| − | color: #a697b8;
| + | |
| − | } | + | |
| | | | |
| − | @media (max-width: 575px) {
| + | .navbar .navbar-brand { |
| − | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item {
| + | color: #edf2f4; |
| − | color: #edf2f4;
| + | } |
| − | }
| + | |
| − | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item:hover,
| + | |
| − | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item:focus {
| + | |
| − | color: #a697b8; | + | |
| − | }
| + | |
| − | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8;
| + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | } | + | |
| | | | |
| − | @media (max-width: 767px) {
| + | .navbar .navbar-brand:hover, |
| − | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item {
| + | .navbar .navbar-brand:focus { |
| − | color: #edf2f4; | + | color: #a697b8; |
| − | }
| + | } |
| − | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item:hover,
| + | |
| − | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item:focus {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8; | + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | } | + | |
| | | | |
| − | @media (max-width: 991px) {
| + | .navbar .navbar-text { |
| − | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item {
| + | color: #edf2f4; |
| − | color: #edf2f4;
| + | } |
| − | }
| + | |
| − | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item:hover,
| + | |
| − | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item:focus {
| + | |
| − | color: #a697b8; | + | |
| − | }
| + | |
| − | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8;
| + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | } | + | |
| | | | |
| − | @media (max-width: 1199px) {
| + | .navbar .navbar-text a { |
| − | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item {
| + | color: #a697b8; |
| − | color: #edf2f4; | + | } |
| − | }
| + | |
| − | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item:hover,
| + | |
| − | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item:focus {
| + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8; | + | |
| − | background-color: #8e44ad;
| + | |
| − | }
| + | |
| − | } | + | |
| | | | |
| − | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item { | + | .navbar .navbar-text a:hover, |
| − | color: #edf2f4;
| + | .navbar .navbar-text a:focus { |
| − | }
| + | color: #a697b8; |
| − | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item:hover,
| + | } |
| − | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item:focus { | + | |
| − | color: #a697b8;
| + | |
| − | }
| + | |
| − | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item.active {
| + | |
| − | color: #a697b8;
| + | |
| − | background-color: #8e44ad;
| + | |
| − | } | + | |
| | | | |
| − | footer.nb-footer {
| + | .navbar .navbar-nav .nav-link { |
| − | background: #222;
| + | color: #edf2f4; |
| − | border-top: 4px solid #8e44ad; }
| + | border-radius: .25rem; |
| − | footer.nb-footer .about {
| + | margin: 0 0.25em; |
| − | margin: 0 auto;
| + | } |
| − | margin-top: 40px;
| + | |
| − | max-width: 1170px;
| + | |
| − | text-align: center; }
| + | |
| − | footer.nb-footer .about p {
| + | |
| − | font-size: 13px;
| + | |
| − | color: #999; | + | |
| − | margin-top: 30px; }
| + | |
| − | footer.nb-footer .about .social-media {
| + | |
| − | margin-top: 15px; }
| + | |
| − | footer.nb-footer .about .social-media ul li a {
| + | |
| − | display: inline-block;
| + | |
| − | width: 45px;
| + | |
| − | height: 45px;
| + | |
| − | line-height: 45px;
| + | |
| − | border-radius: 50%; | + | |
| − | font-size: 16px;
| + | |
| − | color: #9a24c2;
| + | |
| − | border: 1px solid rgba(255, 255, 255, 0.3); }
| + | |
| − | footer.nb-footer .about .social-media ul li a:hover {
| + | |
| − | background: #8e44ad;
| + | |
| − | color: #fff;
| + | |
| − | border-color: #8e44ad; }
| + | |
| − | footer.nb-footer .footer-info-single {
| + | |
| − | margin-top: 30px; } | + | |
| − | footer.nb-footer .footer-info-single .title {
| + | |
| − | color: #aaa;
| + | |
| − | text-transform: uppercase;
| + | |
| − | font-size: 16px;
| + | |
| − | border-left: 4px solid #8e44ad;
| + | |
| − | padding-left: 5px; }
| + | |
| − | footer.nb-footer .footer-info-single ul li a {
| + | |
| − | display: block;
| + | |
| − | color: #aaa;
| + | |
| − | padding: 2px 0; }
| + | |
| − | footer.nb-footer .footer-info-single ul li a:hover {
| + | |
| − | color: #8e44ad; }
| + | |
| − | footer.nb-footer .footer-info-single p {
| + | |
| − | font-size: 13px;
| + | |
| − | line-height: 20px;
| + | |
| − | color: #aaa; }
| + | |
| − | footer.nb-footer .copyright {
| + | |
| − | margin-top: 15px;
| + | |
| − | background: #111;
| + | |
| − | padding: 7px 0;
| + | |
| − | color: #999; }
| + | |
| − | footer.nb-footer .copyright p {
| + | |
| − | margin: 0;
| + | |
| − | padding: 0; }
| + | |
| − | div[class="footer-info-single"]>ul{
| + | |
| − | margin: 0;
| + | |
| − | }
| + | |
| − | div[class="footer-info-single"]>h2{
| + | |
| − | padding-bottom: 0;
| + | |
| − | padding-top: 0;
| + | |
| | | | |
| − | margin-bottom: .5rem;
| + | .navbar .navbar-nav .nav-link:not(.disabled):hover, |
| − | } | + | .navbar .navbar-nav .nav-link:not(.disabled):focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar .navbar-nav .dropdown-menu { |
| | + | background-color: #151616b6; |
| | + | border-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-nav .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar .navbar-nav .dropdown-menu .dropdown-item:hover, |
| | + | .navbar .navbar-nav .dropdown-menu .dropdown-item:focus, |
| | + | .navbar .navbar-nav .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-nav .dropdown-menu .dropdown-divider { |
| | + | border-top-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-nav .nav-item.active .nav-link, |
| | + | .navbar .navbar-nav .nav-item.active .nav-link:hover, |
| | + | .navbar .navbar-nav .nav-item.active .nav-link:focus, |
| | + | .navbar .navbar-nav .nav-item.show .nav-link, |
| | + | .navbar .navbar-nav .nav-item.show .nav-link:hover, |
| | + | .navbar .navbar-nav .nav-item.show .nav-link:focus { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-toggler { |
| | + | border-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-toggler:hover, |
| | + | .navbar .navbar-toggler:focus { |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | |
| | + | .navbar .navbar-toggler .navbar-toggler-icon { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar .navbar-collapse, |
| | + | .navbar .navbar-form { |
| | + | border-color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar .navbar-link { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar .navbar-link:hover { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | @media (max-width: 575px) { |
| | + | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item:hover, |
| | + | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item:focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar-expand-sm .navbar-nav .show .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | } |
| | + | |
| | + | @media (max-width: 767px) { |
| | + | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item:hover, |
| | + | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item:focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar-expand-md .navbar-nav .show .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | } |
| | + | |
| | + | @media (max-width: 991px) { |
| | + | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item:hover, |
| | + | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item:focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar-expand-lg .navbar-nav .show .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | } |
| | + | |
| | + | @media (max-width: 1199px) { |
| | + | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item:hover, |
| | + | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item:focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar-expand-xl .navbar-nav .show .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | } |
| | + | |
| | + | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item { |
| | + | color: #edf2f4; |
| | + | } |
| | + | |
| | + | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item:hover, |
| | + | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item:focus { |
| | + | color: #a697b8; |
| | + | } |
| | + | |
| | + | .navbar-expand .navbar-nav .show .dropdown-menu .dropdown-item.active { |
| | + | color: #a697b8; |
| | + | background-color: #8e44ad; |
| | + | } |
| | + | |
| | + | footer.nb-footer { |
| | + | background: #222; |
| | + | border-top: 4px solid #8e44ad; |
| | + | } |
| | + | |
| | + | footer.nb-footer .about { |
| | + | margin: 0 auto; |
| | + | margin-top: 40px; |
| | + | max-width: 1170px; |
| | + | text-align: center; |
| | + | } |
| | + | |
| | + | footer.nb-footer .about p { |
| | + | font-size: 13px; |
| | + | color: #999; |
| | + | margin-top: 30px; |
| | + | } |
| | + | |
| | + | footer.nb-footer .about .social-media { |
| | + | margin-top: 15px; |
| | + | } |
| | + | |
| | + | footer.nb-footer .about .social-media ul li a { |
| | + | display: inline-block; |
| | + | width: 45px; |
| | + | height: 45px; |
| | + | line-height: 45px; |
| | + | border-radius: 50%; |
| | + | font-size: 16px; |
| | + | color: #9a24c2; |
| | + | border: 1px solid rgba(255, 255, 255, 0.3); |
| | + | } |
| | + | |
| | + | footer.nb-footer .about .social-media ul li a:hover { |
| | + | background: #8e44ad; |
| | + | color: #fff; |
| | + | border-color: #8e44ad; |
| | + | } |
| | + | |
| | + | footer.nb-footer .footer-info-single { |
| | + | margin-top: 30px; |
| | + | } |
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| | + | footer.nb-footer .copyright p { |
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| | + | div[class="footer-info-single"]>ul { |
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| | <a class="dropdown-item" href="https://2021.igem.org/Team:Qdai/Partnership">Partnership</a> | | <a class="dropdown-item" href="https://2021.igem.org/Team:Qdai/Partnership">Partnership</a> |
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| − | <a class="dropdown-item" href="https://2021.igem.org/Team:Qdai/Another_area">Anorher Area</a> | + | <a class="dropdown-item" href="https://2021.igem.org/Team:Qdai/Another_area">Another Area</a> |
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| Line 387: |
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| | <div class="bg" style="margin-top: -130px;"> | | <div class="bg" style="margin-top: -130px;"> |
| − | <img alt="" class="top-text2" src="https://static.igem.org/mediawiki/2021/e/ed/T--Qdai--img--We_are_iGEM_Qdai.png" style="top:10vw;"/> | + | <img alt="" class="top-text2" src="https://static.igem.org/mediawiki/2021/e/ed/T--Qdai--img--We_are_iGEM_Qdai.png" style="top:10vw;"/> |
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| | <div class="selection" style="height: auto;"> | | <div class="selection" style="height: auto;"> |
| | <div class="container-vcenter"> | | <div class="container-vcenter"> |
| | <h2 class="tag"><span>Experiments</span></h2> | | <h2 class="tag"><span>Experiments</span></h2> |
| | + | <embed src="https://static.igem.org/mediawiki/2021/a/a6/T--Qdai--img--Experiments.docx.pdf" style="height: 100vh;" type="application/pdf" width="80%"/> |
| | | | |
| − | <div class="main" style="width: 60%;margin-bottom: 8vh;">
| + | |
| − | <h2 class="sub">Preparation of TE Buffer
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Tris-HCl: 2ml <br/>
| + | |
| − | ・EDTA: 0.5ml <br/>
| + | |
| − | ・dH2O: 197.5ml <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Mix the above materials. <br/>
| + | |
| − | 2. Place the mixture in a medium bottle and cover with aluminum foil <br/>
| + | |
| − | 3. Autoclave. Following autoclave, store it at room temperature.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Preparation of LB medium</h2>
| + | |
| − | <p>
| + | |
| − | Materials <br/>
| + | |
| − | ・Tryptone: 10g <br/>
| + | |
| − | ・Yeast Extract: 5g <br/>
| + | |
| − | ・NaCl: 5g <br/>
| + | |
| − | ・dH2O: 1000ml <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Put the above materials into a beaker, cover with aluminum foil, and mix with a stirrer. <br/>
| + | |
| − | 2. Autoclave. Following autoclave, store it in a medium bottle at 4 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Preparation of Ampicillin
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Ampicillin sodium salt: 0.5g <br/>
| + | |
| − | ・Sterile water: 5ml <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Mix the above materials and vortex. <br/>
| + | |
| − | 2. Filter the mixture through a syringe filter. <br/>
| + | |
| − | 3. Dispense the amount to be used into a tube and store at -20 ℃. (If you use 50% ethanol instead of sterile water, you do not need to dispense.)
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Preparation of LB Agar Plates
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Tryptone: 4g <br/>
| + | |
| − | ・Yeast Extract: 2g <br/>
| + | |
| − | ・NaCl: 2g <br/>
| + | |
| − | ・Agar (not agarose): 6g <br/>
| + | |
| − | ・Antibiotic (ampicillin): 400㎕ <br/>
| + | |
| − | ・dH2O: 400ml <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Put Tryptone, Yeast Extract, NaCl, and dH2O into a beaker, cover with aluminum foil, and mix with a stirrer. <br/>
| + | |
| − | 2. Add agar and autoclave. <br/>
| + | |
| − | 3. Mix with a stirrer. <br/>
| + | |
| − | 4. When the solution cools to about 60 ℃, add ampicillin and mix. <br/>
| + | |
| − | 5. Transfer the mixture to a Petri dish on a clean bench. When the agar has set, stack the Petri dishes, cover them with plastic wrap, and store them at 4 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Resuspending gBlocks® Gene Fragments (IDT)
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・DNA <br/>
| + | |
| − | ・TE Buffer <br/>
| + | |
| − |
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Prior to opening, centrifuge the tube at a minimum of 3000×g to ensure that the material is at the bottom of the tube <br/>
| + | |
| − | 2. Add TE to reach a final concentration of 10ng/㎕. <br/>
| + | |
| − | 3. Vortex briefly <br/>
| + | |
| − | 4. Incubate at 50 ℃ for 20 minutes. <br/>
| + | |
| − | 5. Briefly vortex and centrifuge.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Gibson Assembly (NEB)
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・DNA <br/>
| + | |
| − | ・Gibson Assembly® Master Mix (E2611) <br/>
| + | |
| − | ・Sterile water <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Determine the amount of each material according to the table below. <br/>
| + | |
| − | <img alt="" src="https://static.igem.org/mediawiki/2021/2/27/T--Qdai--img--exp--1.png"/> <br/>
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − | 2. Add sterile water, DNA, and Gibson Assembly Master Mix to the microtube in that order. <br/>
| + | |
| − | 3. Incubate samples in a thermocycler at 50 ℃ for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples at -20 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">DNA purification
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・DNA <br/>
| + | |
| − | ・Wizard SV® Gel and PCR Clean-up System (Promega) <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Add an equal volume of Membrane Binding Solution to DNA. <br/>
| + | |
| − | 2. Insert SV Minicolumn into Collection Tube. <br/>
| + | |
| − | 3. Transfer DNA to the Minicolumn assembly. Incubate at room temperature for 1 minute. <br/>
| + | |
| − | 4. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. <br/>
| + | |
| − | 5. Add 700㎕ Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. <br/>
| + | |
| − | 6. Repeat Step 5 with 500㎕ Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes. <br/>
| + | |
| − | 7. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol. <br/>
| + | |
| − | 8. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. <br/>
| + | |
| − | 9. Add 50㎕ of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. <br/>
| + | |
| − | 10. Discard Minicolumn and store DNA at 4℃ or –20℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Dilution of TAE Buffer (10×)
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・TAE Buffer(10×): 100ml <br/>
| + | |
| − | ・MiliQ: 900ml <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Mix the above materials and store them in a medium bottle at room temperature.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Preparation of agarose gel
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・TAE Buffer: 20ml <br/>
| + | |
| − | ・Agarose: 0.2㎕ <br/>
| + | |
| − | ・Midori Green: 1㎕ <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Put TAE Buffer and Agarose in a beaker and cover with plastic wrap. <br/>
| + | |
| − | 2. Melt the Agarose in a microwave oven. <br/>
| + | |
| − | 3. When the solution is cool enough not to be hot to the touch, add the Midori Green. <br/>
| + | |
| − | 4. Pour the gel into the casting stand and set the comb. <br/>
| + | |
| − | 5. When the gel hardens, cover with plastic wrap and store at 4 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Agarose Gel Electrophoresis
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・DNA: 1㎕ of each <br/>
| + | |
| − | ・DNA Ladder: 5㎕ <br/>
| + | |
| − | ・Loading Dye (6×): 1㎕×(n+1) <br/>
| + | |
| − | ・Sterile water: 4㎕×n <br/>
| + | |
| − | ・TAE Buffer <br/>
| + | |
| − | (n: Number of samples) <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Add sterile water4㎕, Loading Dye (6×)1㎕, and DNA1㎕to the PCR tube. <br/>
| + | |
| − | 2. Add DNA Ladder5㎕ and Loading Dye (6×)1㎕to the PCR tube. <br/>
| + | |
| − | 3. Place the agarose gel into the gel box. <br/>
| + | |
| − | 4. Fill the gel box with TAE Buffer until the gel is covered. <br/>
| + | |
| − | 5. Loading the samples. <br/>
| + | |
| − | 6. Run the gel at 100V for 15-25 minutes. <br/>
| + | |
| − | 7. Check the bands by UV irradiation.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Digestion</h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Restriction enzyme <br/>
| + | |
| − | (EcoRⅠ-HF, HindⅢ) <br/>
| + | |
| − | ・NEB Buffer r2.1 (10×) <br/>
| + | |
| − | ・DNA <br/>
| + | |
| − | ・Sterile water <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Determine the amount of each material according to the table below. <br/>
| + | |
| − | <img alt="" src="https://static.igem.org/mediawiki/2021/0/0d/T--Qdai--img--exp--2.png"/> <br/>
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − | 2. Add sterile water, Buffer, restriction enzyme, and DNA to the microtube in that order. <br/>
| + | |
| − | 3. Incubate samples in a thermocycler at 37 ℃ for 1 hour. Following incubation, store samples at -20 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Ligation
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Ligation Mix: 3㎕ <br/>
| + | |
| − | ・Sterile water: 18㎕ <br/>
| + | |
| − | ・DNA <br/>
| + | |
| − |
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Prepare Mighty Mix by mixing Ligation Mix and sterile water. <br/>
| + | |
| − | 2. Determine the amount of each material according to the table below. <br/>
| + | |
| − | <img alt="" src="https://static.igem.org/mediawiki/2021/1/18/T--Qdai--img--exp--3.png" style="height: 10vh;"/> <br/>
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − |
| + | |
| − | 3. Add vector, insert, and Mighty Mix to the microtube. (For negative controls, add TE Buffer instead of Insert.) <br/>
| + | |
| − | 4. Incubate samples in a thermocycler at 16 ℃ for 30 minutes. Following incubation, store samples at -20 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Transformation
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Plasmid: 6㎕ of each (Recombinant plasmid and Negative Control) <br/>
| + | |
| − | ・Competent cells: 50㎕ ×n <br/>
| + | |
| − | ・SOC medium (or LB medium) 1ml×n <br/>
| + | |
| − | ・LB Agar Plates: 2 Plates ×n <br/>
| + | |
| − | (n: Number of samples) <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Add plasmid and competent cells to the microtubes. <br/>
| + | |
| − | 2. Place the microtubes on ice for 30 minutes. <br/>
| + | |
| − | 3. Heat shock at 42 ℃ for 45 seconds <br/>
| + | |
| − | 4. Add SOC medium to the microtubes. <br/>
| + | |
| − | 5. Place the microtubes at 37 ℃ for 30 minutes. <br/>
| + | |
| − | 6. Spread 100㎕ onto the plate. <br/>
| + | |
| − | 7. Centrifuge the rest of the mixture at 27°C, 3000 rpm for 1 minute. <br/>
| + | |
| − | 8. Discard about 700㎕ of the supernatant. <br/>
| + | |
| − | 9. Dissolve the precipitate by pipetting slowly and spread 200㎕ onto the plate. <br/>
| + | |
| − | 10. Incubate at 37℃ for 14~18 hours. Following incubation, cover samples in plastic wrap and store them at 4 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Inoculation</h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・LB medium: 3ml×n <br/>
| + | |
| − | ・Antibiotic (ampicillin): 3㎕×n <br/>
| + | |
| − | ・LB Agar Plates with colony <br/>
| + | |
| − | (n: Number of samples) <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Mix LB medium and ampicillin. <br/>
| + | |
| − | 2. Dispense 3ml into each culture tube. <br/>
| + | |
| − | 3. Pick the colonies from LB Agar Plates using a yellow tip and eject them into a culture tube. <br/>
| + | |
| − | 4. Incubate at 37 ℃ for 12~16 hours in a shaking incubator.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Miniprep (Promega)
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・Bacterial culture 2.4ml×n <br/>
| + | |
| − | ・PureYieldTM Plasmid Miniprep System <br/>
| + | |
| − | (n: Number of samples) <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Centrifuge 1.2ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. <br/>
| + | |
| − | 2. Discard the supernatant. <br/>
| + | |
| − | 3. Add an additional 1.2ml of bacterial culture to the same tube. Repeat Steps 1 and 2. <br/>
| + | |
| − | 4. Add 600㎕ of TE buffer or water to the cell pellet, and resuspend completely. <br/>
| + | |
| − | 5. Add 100㎕ of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times. <br/>
| + | |
| − | 6. Add 350㎕ of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting. <br/>
| + | |
| − | 7. Centrifuge at maximum speed in a microcentrifuge for 3 minutes. <br/>
| + | |
| − | 8. Transfer the supernatant (~900㎕) to a PureYield™ Minicolumn without disturbing the cell debris pellet. <br/>
| + | |
| − | 9. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds. <br/>
| + | |
| − | 10. Discard the flowthrough, and place the minicolumn into the same Collection Tube. <br/>
| + | |
| − | 11. Add 200㎕ of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. <br/>
| + | |
| − | 12. Add 400㎕ of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds. <br/>
| + | |
| − | 13. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30㎕ of Elution Buffer or nuclease-free water directly to the minicolumn matrix. Let stand for 1 minute at room temperature. <br/>
| + | |
| − | 14. Centrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20 ℃.
| + | |
| − | </p>
| + | |
| − | <h2 class="sub">Measurement of DNA concentration (NanoDrop)
| + | |
| − | </h2>
| + | |
| − | <p>Materials <br/>
| + | |
| − | ・DNA 1.5㎕ <br/>
| + | |
| − | ・Buffer 1.5㎕ <br/>
| + | |
| − | Protocol <br/>
| + | |
| − | 1. Start nanodrop2000 <br/>
| + | |
| − | 2. Perform calibration and blank measurement by entering one drop of 1.5㎕ buffer. <br/>
| + | |
| − | 3. Clean the surface of the NanoDrop with KimWipes. <br/>
| + | |
| − | 4. place 1.5㎕ per sample on the NanoDrop and measure the concentration.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 709: |
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