background-image: url('https://static.igem.org/mediawiki/2021/f/f8/Small_icon_ambassadors.svg'); background-color: #fff; } .two_third_size { } $(".current_hub .igem_submenu").prev().toggleClass("showing"); background-color: #2C3E66 !important; Before setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector to use for the ligation reaction. //sponsors $("#" + destination_div).load(source_page, function() {}); break; .row > .column.three_quarter_size > .highlight.gray { } You can also try other ratios (1:1, 2:1, and up to 7:1) if the 3:1 fails to yield good results. ZmPHO2 expression as determined by RT-qPCR in miR399-OE and miR399-STTM transgene-positive lines and the corresponding negative controls. $("ul.image_slider li.current_image").removeClass("current_image"); z-index: 100; .left_square.ambassadors_icon{ } background-color:#2C3E66; } border: 1px solid #2C3E66; } } .igem_column_wrapper { height: 28px; ]; Editing, Cloning height: 0; } Mix thoroughly and incubate at 16 C . } } letter-spacing: 0.25px; font-weight:bold; background-color: #eae7e5; } //filtering options a.image_href{ Collaborations //make clicked element have a temp class //make months + days look nice $(button + '.temp').removeClass('temp').addClass('current'); $('#scroll_list_display').click(function() { $("#HQ_page").attr('id', ''); I strongly urge you to always run controls for these reactions as they will give you a lot of information and allow you to more easily troubleshoot your cloning problems. } case 2: } } var index = gallery_ids.indexOf(clicked_id); p.center_content, h1.center_content, h2.center_content, h3.center_content, h4.center_content, h5.center_content, h6.center_content, img.center_content { border: 2px solid #99948f; .full_size { content:""; $('.current_option').removeClass('current_option'); position: relative; New England Biolabs has a useful online Ligation Calculator that can help you determine how much of your insert DNA you need to add in various gram values. content:""; /*coming soon button */ height: 25px; background-size: 25px 25px; /* 25% */ font-weight: bold; .highlight.post > .details > .contact::before { Addgene: Protocol - How to Ligate Plasmid DNA /*menu item styling // layer one*/ var selected_sorting_type; background-size: 25px 25px; } /**********************************************************************************************/ }, 5000); .half_size { While you may think this means your reaction worked really well, it actually indicates a problem with your restriction digest. Positive control for transformation: transformation of an intact vector with ampicillin resistance at a defined concentration (e.g. //append the options float: left; background-size: 25px; width:100%; background-image: url('https://static.igem.org/mediawiki/2021/7/7c/Small_icon_igem_gear.svg'); /*double the extra space to page break with clear class*/ /*width for highlight boxes in rows*/ //get minutes background-color: #5C6F9B!important; background-color:white; Introduction, .submenu_item.subsection_title{ } font-family:Arial; } padding: 9px 3% 2px 3%; } margin-bottom: 0px; border-radius:5px; /*submenu item styling // layer three*/ outline: 1px solid #e2dee1; border: 4px solid #2C3E66; .column.full_size > .highlight.post { //once timezone is ready, allow user to change time zones height: 25px; text-align: center; $(slide + '.current').removeClass('current'); min-height: 28px; } Negative and Positive Ligation Controls. "UTC -02", "UTC -01", "UTC 0", "UTC +01", "UTC +02 (CEST)", "UTC +03", } display: flex; float:right; background-image: url('https://static.igem.org/mediawiki/2021/7/73/Safety_enviromentalsamples.svg'); } text-decoration:none; .igem_content_wrapper { background-image: url(https://static.igem.org/mediawiki/2021/f/f5/Collab_contact.svg); .hub_icon.measurement{ } if (wgPageName.includes("Schedule")) { for (y = 0; y < gallery_ids.length; y++) { $('#' + clicked_id + '> .content > .slide').html(function(u) { } time_start = time_start - 2400; .sort_option{ width: 25px; height: 30px; width: 100%; .igem_menu_control_mobile { opacity: 100%; .igem_subsubmenu{ After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation. if ($(this).hasClass("next")) { float:left; text-decoration:none; Chemically competent cells are treated with a buffer that contains CaCl2 and other saltsa process that is thought to create pores in the bacterial membrane, as well as mask the DNA's negative charge and promote its binding to the cell membrane. background-color: inherit; $(this).addClass('temp'); #filtered_options{ } margin-bottom:5px; //hide / show content controller_number = $("ul.image_slider li.current_image").index() + 1; /*image caption*/ font-family: Freeroad_Bold, Arial; } } } DNA ligation is no different. background-color: #2c3e66; float:left; case "pauljames": right: 0; border-top: 1px solid #99948f; //get which gallery was clicked } How do I place an order? /* iconography for posts */ font-weight: bold; margin-bottom: -5px; }); //clean and remove } background-color: #C14998; width:62%; .igem_menu_logo { } .accordion_control.purple{ case "hassnain_qasim": //time zones margin-bottom: -5px; case "jakebeal": content: ""; var date_changed = month_day_for_display(month, day) + ", " + background-image: url(https://static.igem.org/mediawiki/2021/e/e5/Competition_deliverables_titleandabstract.svg); margin-right: 5px; .scrolling_list.show_all::-webkit-scrollbar-track { /*main layout class */ } if (type_of_click == 'navigation') { const months_array = ['January', 'February', 'March', 'April', 'May', 'June', .highlight.border.teal { Positive reaction. background-color:#2C3E66; padding-bottom: 0px; border: 4px solid #111c4e; .calendar_accordion_content{ /******************************************************************************************/ /******************************************************************************************/ .gallery > .controls > .button{ $('.date_timezone').each(function(i, obj) { background-image: url(https://static.igem.org/mediawiki/2021/f/f5/Collab_contact.svg); } minutes: 0, //switch hours from 24 to 12 format margin-bottom: -5px; font-size: 18px; color: #2d3e66; background-color: #F69C55 !important; Solved 2. Last week, we discussed two negative controls for - Chegg //time zone functions } if (hour_x > 1159) { float: left; //this will hold the options to append to the dropdown select text-align:right; case "alexanian": } .column.full_size > .highlight, .column.half_size > .highlight, .column.third_size > .highlight, .column.two_third_size > .highlight, .column.three_quarter_size > .highlight, .column.quarter_size > .highlight{ .igem_column_wrapper p.image_caption { //showing a longer list content:""; width: 25px; $(button + '.current').next().addClass('temp'); .accordion_control.expanded::before{ max-width: 100px; padding:0px; .scrolling_list::-webkit-scrollbar { display: inline-block; background-size: 20px; background-color: #e2dee1; height: 35px; float: left; width: 75%; } /* 1100px */ } text-align: center; padding: 7px 15px 0px 15px; background-color: #f8ed6e; background-image: url(https://static.igem.org/mediawiki/2019/d/d2/Menu_icon_measurement.svg); grab_date = $(this).html(); $(this).addClass('current_option'); background-image: url(https://static.igem.org/mediawiki/2021/5/59/Competition_fees.svg); } width: 95%; } height: 15px; /*styled select dropdowns */ }); display: inline-block; .content > .navigation.next{ //accessing submenus and subsubmenus background-color:#ffffff; page_url = "https://2021.igem.org/"; font-size: 22px; duration = (((duration / 60) | 0) * 100) + (duration % 60); border-radius: 5px; font-weight: bold; switch ((wgPageName.match(/\//g) || []).length) { content: ""; .igem_logo_mobile img { hour_obj.minutes = hour_x % 100; background-image: url('https://static.igem.org/mediawiki/2021/3/3e/Small_icon_working_group.svg'); .subtitle_wrapper > .icon.competition_deliverables_judging_form{ display:none; //display what option the user selected The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration. case "Bluepumpkin": } It's easy to forget or skip controls when you're doing restriction digests and ligations. Sitemap, Resources/Troubleshooting/Restriction Digests and Ligations, Colonies will give you an idea of the background due to uncut vector due to inefficient restriction digest of the backbone, Colonies will give you an idea of the background due to the re-circularization of cut backbone, Colonies indicate contamination of intact/uncut plasmid in your ligation and/or transformation reagents, the length of your Vector DNA (or backbone). } $(".calendar_accordion_control").click(function() { color: black; height: 36px; This page is focused on common problems researchers have with ligations and restriction digests. } hold_all_dates[x][3], margin-top: 5px; //note that, if duration is already in military format, this makes no change background-repeat: no-repeat; background-image: url(https://static.igem.org/mediawiki/2021/f/ff/Competition_deliverables_wiki.svg); .accordion_control:hover, .accordion_control.purple:hover{ } Receive the latest news, hot plasmids, discounts and more. padding-left: 20px; Now that you know your transformation efficiency and it's above 1 x 108 CFU/g DNA, we can work on other possible problems if you're still not getting great results from your cloning. color: white!important; } margin-top: -7px; width: 1em; .left_square.igemhq_icon{ } /* MENU */ case "Kitwa":

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