background-size: 150px; float:right; $("#HQ_page").attr('id', ''); .highlight.post > .details > .title.survey::before { .igem_content_wrapper.dark p, .igem_content_wrapper.dark h1, .igem_content_wrapper.dark h2, .igem_content_wrapper.dark h3, .igem_content_wrapper.dark h4, .igem_content_wrapper.dark h5, .igem_content_wrapper.dark h6, .igem_content_wrapper.dark > .igem_column_wrapper > .column.full_size > .highlight.post > .details { Ligation Troubleshooting. Calendar background-image: url('https://static.igem.org/mediawiki/2021/5/51/Small_icon_why.svg'); After. $('.date_timezone').each(function(i, obj) { //convert duration minutes in military format (e.g. float: left; } .image_href:hover{ .submenu_access { case "drew": width: 25px; height: 33px; }); if (day > months_days_limit) { } content: ""; //is it previous or next? color: #000000; Due to COVID-19, iGEM staff are working remotely and cannot receive phone calls. background-color: #fff; background-size: 25px 25px; } .igem_column_wrapper h1, .igem_column_wrapper h2 {
Restriction Digests and Ligations - 2019.igem.org } /**gallerys *****************************************************************************/ case "jakebeal": /* text */ } } //if there is a gallery/carousels on the page } }); .subtitle_wrapper > .icon.judging_medals{ text-decoration: underline; // This is the jquery part of your template. width: 30px; max-width: 300px; background-image: url(https://static.igem.org/mediawiki/2021/9/95/Competition_deliverables_judgingform.svg); var index = gallery_ids.indexOf(clicked_id); height: 25px; .igem_content_wrapper { background-color: #2C3E66; } } case "THaddock": content:""; } else { background-image: url(https://static.igem.org/mediawiki/2021/5/50/Judging_rubric3.png); background-color: #f8ed6e; padding-left: 5%; /*when the submenu item has a subsubmenu - make room for submenu access class on the same line*/ Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. margin-bottom: 0px; text-decoration:none; case "Meagan": controller_number -= 1; $(".current_hub .igem_submenu").fadeToggle(400); "UTC_plus_04", "UTC_plus_05", "UTC_plus_06", "UTC_plus_07", "UTC_plus_08", "UTC_plus_09", bottom: 25%; Learn more, Download our file to copy and paste plasmid data, Learn more about Addgene materials from user-contributed reports describing AAV and antibody experiments, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. font-weight: bold; .red_text { } background-image: url(https://static.igem.org/mediawiki/2021/6/6d/Competition_deliverables_safetyforms.svg); .gallery > .controls > .button{ } Use a ligation calculator to easily quantify how much vector and insert DNA to use. Importantly, this method does not use the Positive Control or Positive Ligation Control probes for any of these calculations. if ($("#load_news_here").length) { $('#' + clicked_id + '> .content > .slide').html(function(u) { float: right; .gallery > .content > .slide{ width:40px!important; $("ul.image_number li:nth-child(" + clicked_image_number + ")").addClass("current_image_number"); .igem_logo_mobile img { background-image: url(https://static.igem.org/mediawiki/2021/6/64/Collab_location.svg); overflow-y: scroll; background-image: url(https://static.igem.org/mediawiki/2019/d/d2/Menu_icon_measurement.svg); @font-face { .subtitle_wrapper > .icon.competition_deliverables_track_selection{ border-bottom: 1px solid #99948f; content:"+"; } $('.gallery:eq(' + x + ') > .content').prepend(navigation); border: 4px solid #99948F; background-color:#ed242724; /*deadline's date*/ // load desired content function width:100%; case "alexanian": .hub_icon > .new_content{ How do I prepare and deposit my plasmids? } //news items } switch ((wgPageName.match(/\//g) || []).length) { var page_url = "https://2021.igem.org/"; background-color:#00afaa; width: 2%; .calendar_accordion_control{ .subtitle_wrapper > .icon.safety_whitelist{ .third_size { /*40px*/ $(this).toggleClass("hide"); width:100%; font-size: 110%; display: inline-block; color:#F59B55; background-image: url(https://static.igem.org/mediawiki/2021/b/b9/Competition_register_team.svg); height: 25px; .column.third_size > .highlight{ content: "Show less"; border: 4px solid #F9EE6E; .highlight.dark{ Negative control = destination vector that is cut and dephorphorylated (if doing non directional cloning that goes into ligation reactionThe positive control will tell you if your transformation is working i.e. $(".with_subsub_menu.current_page").parent().next().toggleClass("showing"); } } .subtitle_wrapper > .accordion{ .igem_column_wrapper a:focus { if (time_end_minutes == 60) { /*Make text red and bold*/ $(this).parent().parent().next().toggleClass("open"); content:""; } color: #009f98; color: #000000; "
.content> .navigation').click(function() { /*video containers and iframes */ line-height: initial; Troubleshooting DNA Ligation Problems - Bitesize Bio width:75%; If you run into any problems registering, depositing, or ordering please contact us at [emailprotected]
.highlight.border.wine { } /******************************************/ //time zones margin-right: 8px; .deadline_content { background-image: url('https://static.igem.org/mediawiki/2021/a/a8/Small_icon_notice.svg'); time_end = (time_start - (time_start % 100)) + time_end_minutes; letter-spacing: 0.25px; //from those minutes, get how many hours .subtitle_wrapper > .icon.competition_deliverables_title_abstract{ } display:block; min-height: 28px; } .highlight.post > .details > .title.working_group::before { } height: 15px; //remove that id from rotation height: 25px; function month_day_for_display(month, day) { padding: 2%; case "Bluepumpkin": /* iconography for posts */ body { //for next slide, add current status so it can be seen now } opacity: 100%; hold_content = ""; function menu_functionality() { /*image opacity */ color:#ffffff; border-bottom: 1px solid #99948f; //read more functionality background-size: 25px 25px; background-image: url(https://static.igem.org/mediawiki/2019/7/73/Menu_icon_sponsors.svg); } font-style: italic; padding: 0px 5% 0px 1%; background-color: #ddd6d0; margin-right: 1%; background-color: white; }); width: 25px; display:none; background-position: center; content:""; background-size: 25px; You should never assume that your digest worked as expected. $('.current_option').removeClass('current_option'); text-align: center; .subtitle_wrapper > .icon.competition_deliverables_presentation{ text-decoration:none; display:none; $(".igem_content_wrapper").hide(); border: 2px solid #2C3E66; width: 36px; /******************************************************************************************/ Cloning blues, why do I have more colonies on negative control? gallery_ids[x] = $('.gallery.carousel:eq(' + x + ')').attr('id'); }); clicked_gallery = $(this).parent().parent().attr('id'); What do I need to know about the customs and importation process for my country? /* 25% */ font-family: Freeroad_Regular; //this is a hub $(".igem_menu").hide(); //hide / show content var gallery_ids = []; //time zone functions padding-left: 20px; color:#2C3E66 !important; } //this will hold the options to append to the dropdown select background-color:#fff; #content { case "Vinoo": .three_quarter_size { DNA ligation is no different. background-image: url(https://static.igem.org/mediawiki/2021/a/a8/Icon_arrow_left.svg); } } case "Nemanja": controller_number = $('ul.image_slider li').length; background-image: url(https://static.igem.org/mediawiki/2021/b/b0/Judging_medals.svg); position: relative; background-color:#f2f2f2; background-color: #F9EE6E !important; width: 15%; padding: 4% 1.5%; background-image: url('https://static.igem.org/mediawiki/2021/3/37/Small_icon_flag.svg'); float: left; case "schlander": } .subsubmenu_item:hover, .igem_submenu > .submenu_access.showing:hover, .submenu_access.showing:hover, .subsubmenu_item.current_subpage { } Cells co-transfected with pbJun-KN151 and pbFos-LC151 plasmids were used as positive control, and those co-transfected with pHA-KN151 and pMyc-LC151 plasmids were used as negative control. page_url = page_url + wgPageName; var selected_sorting_type; display:block; } /***********************************************/ color: #000; hour_obj.hours = switch_to_twelve_hour((hour_x / 100) | 0); background-image: url('https://static.igem.org/mediawiki/2021/c/cb/Small_icon_note.svg'); } /**********************************************************************************************/ float:left; width:100%; border-radius: 5px; $("a[href$='" + page_url + "'] > .submenu_item").addClass("current_page"); .column.half_size > .highlight { Due diligence when it comes to cloning is well worth the effort, I promise you! overflow-y: auto; } width: 25px; border: 4px solid #2C3E66; display: inline-block; } } -ms-transition: all 0.4s ease; } else if (time_end < 0) { background-size: 50px; } $(this).prev(".read_more_text").fadeToggle(400); 1 a, Supplementary Table S1 . content: ""; } // select sub menu page #home_logo, #sideMenu, #sideMenu, #top_title, .patrollink { /*specification for hub icon title images*/ } border: 3px solid #eae7e5; $(slide + '.temp').removeClass('temp').addClass('current'); "UTC -07", "UTC -06", "UTC -05", "UTC -04 (EDT)", "UTC -03", function time_zone_dropdown() { text-decoration: none !important; } .on_page::after{ background-image: url(https://static.igem.org/mediawiki/2021/c/c8/Judging_introduction2.png); $(this).toggleClass("expanded"); margin-bottom: 0px; */ News if ($("#navigation_support").length) { background-image: url('https://static.igem.org/mediawiki/2021/4/44/Small_icon_alert.svg'); } background-color: white; case "Abigail": max-width: 25px; text-align: center; .subtitle_wrapper > .icon.competition_deliverables_wiki{ } } //create an id for that specific date, we will use this later to append margin: -10px 20px 0px 0px; clear: both; By running a few extra reactions, you can . padding-right:10px; font-weight: bold; console.log(check_if_user_allowed()); .subtitle_wrapper > .icon.judging_applytojudge{ } .hub_icon.measurement{ float:right; Note: Try different vector to insert ratios to optimize the ligation reaction. } color: #000000; } background-color: #EE5757 !important; Negative and Positive Ligation Controls. background-image: url('https://static.igem.org/mediawiki/2021/f/fb/Resources_findinspiration.svg'); } .subtitle_wrapper > .icon.competition_fees{ var sixty = ((time_end_minutes / 60) | 0) * 100; .button.purple a { .button.yellow a { function image_slider_rotation() { background-image: url('https://static.igem.org/mediawiki/2021/1/18/Small_icon_sponsors.svg'); width: 75%; else { //check if the user is in the approved list to view Dev pages and HQ info content height: 25px; background-image: url(https://static.igem.org/mediawiki/2021/c/c9/Competition_deliverables_trackselection.svg); } height: 60px; height: 25px; text-decoration:none; height: 25px; .highlight.post > .details > .title.igem_hq::before { //mobile menu access .submenu_item.with_subsub_menu.with_icon > .icon_submenu{ .video-container iframe { var date_changed = month_day_for_display(month, day) + ", " + border-radius: 5%; //create an array to store them .highlight.post > .details > .location::before { padding:10px; margin-bottom: 15px; /************************************************/ float: left; margin-left: -3px; height: 25px; border: 2px solid #c1c1c1; width: 10%; font-size: 20px; display: none; } color: #aab3ca !important; text-align: left; /************/ } Quantitative in situ proximity ligation assays examining protein Events padding-right:5%; } } background-image: url(https://static.igem.org/mediawiki/2021/f/f5/Collab_contact.svg); //add converted minutes //clean hold content /* div to hold a long list and make it scrollable*/ It is important that the same settings (exposure time, gain, filters used, etc.) height: auto; display: flex; Everyone keeps telling me it is best to use several controls, as well as a positive and negative control but I'm not sure which is which. } hour_obj.hours = (hour_x / 100) | 0; .subtitle_wrapper > .icon.safety_environmental{ color: #00afaa !important; .calendar_accordion_control::before { .scroll_control.show_less::before{ .row > .column.three_quarter_size > .highlight.gray { page_url = page_url + wgPageName.substring(0, wgPageName.indexOf('/')); font-size: 17px; background-color: #f2f2f2; .subtitle_wrapper.page_title > .subtitle{ } .hub_icon.resources{ if (controller_number <= 1) { if (hour % 12 == 0) return 12; // no such thing as 0 hours /*hub icon next to hub title*/ padding: 9px 0px; border-bottom:0px; .subtitle_wrapper > .icon.resources_findinspiration{ margin:auto; width: 198px; .highlight.post > .details > .title.sponsors::before { } PDF Lab 4: Gel Electrophoresis - Vanderbilt UniversityAddgene: Protocol - How to Ligate Plasmid DNA } /* specifications for information details for the collaboration page */ .highlight.post > .details > .title.notice::before { border: 4px solid #2C3E66; Transformation is a key step in DNA cloning. const months_array = ['January', 'February', 'March', 'April', 'May', 'June', However, it should not be more that 1-2% of the total number of colonies on the plate. To confirm performance of the reaction components, prepare a positive control ligation reaction to run concurrently with the experimental reaction. var date_counter = 0; ]; clear:both; border-collapse: collapse; top:0; content: "Team: "; .subtitle_wrapper > .icon.competition_register_team{ .gallery > .controls > .button.current, .gallery > .controls > .button:hover{ c_number++; day = days_in_a_month[month]; .hub_icon.calendar{ background-color: #111c4e; content:'SHOW MORE'; $("#filtered_options").empty(); Please contact us by email. $(".image_controller").click(function() { background-size: 25px 25px; } .igem_column_wrapper table { 30, 30, 30, 31 The Positive and Negative Affect Schedule (PANAS) is a widely used measure for assessing levels of positive and negative affect. height: 100%; width:100%; OEP and OEN represent transgene-positive and transgene-negative plants, respectively, derived from the same transformation event. // DAYLIGHT SAVINGS TIME you should see lots of coloniesThe negative control will tell you if your dephorphorylation is working i.e. } .igem_content_wrapper { background-image: url(https://static.igem.org/mediawiki/2021/c/c8/2021_igem_logo_horizontal.svg); case "Pdmillett": font-size: 120%; }); content:""; This is most often entered as nanograms or micrograms. font-weight: normal!important; } width: 100%; For the negative control ligation mix: vector 3 l (81.6 ng) + ddH2O 5 l + T4 DNA Buffer Ligase 1 l + T4 DNA Ligase 1 l. Plasmid Cloning by Restriction Enzyme Digest, Checks viability of competent cells and verifies the antibiotic resistance of the plasmid, Background due to vector re-circularization - most useful for phosphatase treated vector, Any colonies indicate contamination of intact plasmid in ligation or transformation reagents. margin-bottom: -5px; case "Randy": Substitute the vector DNA and insert DNA with 500 ng (1 L) of Control DNA, and replace 5 L of reaction (water) volume with 24% polyethylene glycol. } $(this).addClass('temp'); height: 25px; .read_more_text{ } It's easy to forget or skip controls when you're doing restriction digests and ligations. padding: 10px; /*wine button */ text-align: left; margin-top: -11px; $("#filtered_options").append(hold_content); /*details for the posts */ border-radius: 14%; $('.gallery:eq(' + x + ') > .controls > .button:eq(0)').addClass('current'); } $("ul.image_number li:nth-child(" + slider_counter + ")").addClass("current_image_number"); font-size: 110%; First a bit of DNA ligation background Ligation reactions fall into two categories, depending whether you are trying to join blunt or sticky DNA. } } /* 850px */ background-color: inherit; border-radius: 50%; //store the current number return date_changed; if (select_is_ready) { menu_functionality(); } width:100%; float: right; $('.sort_option').click(function() { margin-left: -3px; } padding: 0px 12px; .igem_logo_mobile { 90 --> 130) } var slide = ''; case "Nluebke": //navigation support $("ul.image_slider li:nth-child(" + controller_number + ")").addClass("current_image"); background-color: inherit; margin-top: 5px; background-color: #5C6F9B; cursor: pointer; margin-bottom: -5px; "' value='" + utc_value + "'>" + Do controls: When doing ligations you should ALWAYS do a vector alone + ligase control. //if the user wants to change the timezone .track_navigation> img { return hour % 12; @media only screen and (max-width: 650px) { margin-left: -3px; } Controls and Tips for TA cloning - Bitesize Bio } } float: left; //add to global variables width: 100%; letter-spacing: 0.25px; } . background-position: center; //add hours plus minutes in military format } border-bottom: 0px; ]; utc_value = utc_value + 100; float: right; display:none; } font-weight: bold; src: url(https://static.igem.org/mediawiki/2021/9/94/Freeroad_Bold.ttf); How do I place an order? //check if the new day switches months Troubleshooting/Restriction Digest and Ligation - 2015.igem.org background-image: url('https://static.igem.org/mediawiki/2021/3/3e/Small_icon_working_group.svg'); $("ul.image_slider li:nth-child(" + slider_counter + ")").addClass("current_image"); width: 40px; content: ""; width: 25px; font-weight: bold; margin-bottom: -5px; } } Hi, I am new at cloning, and having troubles in finding the desired clone. background-image: url(https://static.igem.org/mediawiki/2021/5/56/Competition_deliverables_projectdescription.svg); In this article, we explain how to set up a ligation reaction with a complete set of controls, and use them to troubleshoot the cause of your ligation problems. While you may think this means your reaction worked really well, it actually indicates a problem with your restriction digest. Ligase is used to make bonds between the insert and backbone covalent. break; $(button + '.current').next().addClass('temp'); font-weight: bold; It is easy to forget or skip controls when you are doing restriction digests and ligations. background-image: url('https://static.igem.org/mediawiki/2021/7/7c/Small_icon_igem_gear.svg'); } .highlight.post > .details{ color: #000000; c_number--; background-color: #eae7e5; } load_these_items("https://2021.igem.org/Registration_updates #updates", "load_registration_updates_here");
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