{"id":32201,"date":"2025-08-19T12:45:53","date_gmt":"2025-08-19T16:45:53","guid":{"rendered":"https:\/\/genomequebec.com\/?post_type=service&#038;p=32201"},"modified":"2026-01-12T14:55:25","modified_gmt":"2026-01-12T19:55:25","slug":"whole-plasmid-and-long-pcr-sequencing","status":"publish","type":"service","link":"https:\/\/genomequebec.com\/en\/technological-services\/centre-dexpertise-et-de-services-2\/instructions\/user-guides\/whole-plasmid-and-long-pcr-sequencing\/","title":{"rendered":"Whole plasmid and long PCR sequencing"},"content":{"rendered":"\n<h2 class=\"wp-block-heading has-text-align-center\" id=\"top-of-page\"><strong>Oxford Nanopore<\/strong> <strong>Technology<\/strong><\/h2>\n\n\n\n<h3 class=\"wp-block-heading has-text-align-center\"><em>User Guide<\/em><\/h3>\n\n\n\n<p>The instructions in this guide must be followed carefully to achieve the best possible results and avoid delays in processing your request.<\/p>\n\n\n\n<p>Samples that do not comply with the recommendations may be rejected without compensation.<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><a href=\"#Starting-Material\" data-type=\"internal\" data-id=\"#Starting-Material\">Starting Material<\/a><\/li>\n\n\n\n<li><a href=\"#Requirements---Quality-and-Quantity\" data-type=\"internal\" data-id=\"#Requirements---Quality-and-Quantity\">Requirements \u2013 Quality and Quantity<\/a>\n<ul class=\"wp-block-list\">\n<li class=\"has-black-color has-text-color has-link-color wp-elements-2d271351998a027e8bca3d722d7bcb28\"><a href=\"#Verification-de-la-concentration\" data-type=\"internal\" data-id=\"#Verification-de-la-concentration\"><\/a><a href=\"#Concentration-Verification\" data-type=\"internal\" data-id=\"#Concentration-Verification\">Concentration verification<\/a><\/li>\n\n\n\n<li class=\"has-black-color has-text-color has-link-color wp-elements-e4d7ac0e5f558cc5e2fd11e84ef3aaef\"><a href=\"#Purety-Verification\" data-type=\"internal\" data-id=\"#Purety-Verification\">Purity Verification<\/a><\/li>\n<\/ul>\n<\/li>\n\n\n\n<li><a href=\"#Required-containers-and-sample-identification\" data-type=\"internal\" data-id=\"#Required-containers-and-sample-identification\">Required Containers and Sample Identification<\/a><\/li>\n\n\n\n<li><a href=\"#Service-Request-Form-and-Sample-Submission\" data-type=\"internal\" data-id=\"#Service-Request-Form-and-Sample-Submission\">Service Request Form and Sample Submission<\/a><\/li>\n\n\n\n<li><a href=\"#Sample-Shipment-Preparation\" data-type=\"internal\" data-id=\"#Sample-Shipment-Preparation\">Sample Shipment Preparation<\/a><\/li>\n\n\n\n<li><a href=\"#Transmission-of-results\" data-type=\"internal\" data-id=\"#Transmission-of-results\">Transmission of results<\/a><\/li>\n<\/ul>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-f1ea74b888da7196577b0d8ad8e41b53\" id=\"Starting-Material\">Starting Material<\/h4>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Plasmid<\/strong><\/th><th><strong>PCR product<\/strong><\/th><\/tr><\/thead><tbody><tr><td>Plasmid DNA circular, double-stranded and less than 25kb. &nbsp;<\/td><td>PCR product more than 1500 bp and less than 25 kb. &nbsp;<\/td><\/tr><tr><td>&#8211; <strong>Recommended extraction kit<\/strong>: QIAGEN kits<br><br>&#8211; <strong>Homemade protocol<\/strong>: an additional purification step with a commercial kit is required. <br><br>Otherwise, sequencing quality may be compromised or fail.<\/td><td> <br>&#8211; PCR products must have been purified by a magnetic bead-based method such as AMPure*. <br><br>*Bead purification service is offered for an additional fee and processing time. Contact <a href=\"https:\/\/genomequebec.com\/en\/contact-us\/\" target=\"_blank\" rel=\"noreferrer noopener\">Client Management Office<\/a> for more information. &nbsp;<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div class=\"wp-block-buttons is-horizontal is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-03627597 wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button\"><a class=\"wp-block-button__link wp-element-button\" href=\"#top-of-page\">Return to top of page<\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-bee3914ac10d732d970b4d8b4d00a42f\" id=\"Requirements---Quality-and-Quantity\">Requirements &#8211; Quality and Quantity<\/h4>\n\n\n\n<p>Samples must be eluted in 10 mM Tris buffer, pH 8.5, or nuclease-free ultrapure water.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Sample type<\/strong><\/th><th><strong>Volume<\/strong><\/th><th><strong>Concentration<\/strong><\/th><th><strong>Quality criteria to be met<\/strong><\/th><th><strong>Recommended verification method<\/strong><\/th><th><strong>Impact on sequencing<\/strong><\/th><\/tr><\/thead><tbody><tr><td>Plasmid<\/td><td>20 \u00b5L &nbsp;<\/td><td>40 ng\/\u00b5L to 80 ng\/\u00b5L &nbsp;<\/td><td>Circular, double-stranded, <strong>intact<\/strong><\/td><td rowspan=\"3\">Size and integrity verified by electrophoresis gel<\/td><td rowspan=\"3\"><strong>Degraded\/fragmented sample<\/strong>: May lead to sequencing failure resulting in lack of consensus due to absence of full reads<\/td><\/tr><tr><td>Purified PCR product<\/td><td>20 \u00b5L &nbsp;<\/td><td>40 ng\/\u00b5L to 80 ng\/\u00b5L &nbsp;<\/td><td rowspan=\"2\">Linear, double-stranded, <strong>intact<\/strong><\/td><\/tr><tr><td>Unpurified PCR product (extra service)*<\/td><td>20 \u00b5L &nbsp;<\/td><td>60 ng\/\u00b5L to 120 ng\/\u00b5L &nbsp;<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p>Low-concentrated samples are often degraded, which significantly affects the quality of the results.<\/p>\n\n\n\n<p>Overly concentrated samples will result in sequencing failure.<\/p>\n\n\n\n<p><strong>It is imperative to dilute the samples to the required concentration or a dilution fee will be charged.<\/strong><\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h6 class=\"wp-block-heading has-text-color has-link-color wp-elements-383970138d770b94131ba1cc494c6676\" id=\"Concentration-Verification\">Concentration Verification<\/h6>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Recommended method<\/strong>: Qubit or equivalent fluorometric method.<\/li>\n<\/ul>\n\n\n\n<p><strong>Note<\/strong>: <strong>Spectrophotometric methods<\/strong> such as <strong>Nanodrop<\/strong> are <strong>not recommended<\/strong> for measuring DNA concentration because they tend to overestimate the DNA concentration, particularly for low-concentration samples or those obtained from complex extractions.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h6 class=\"wp-block-heading has-text-color has-link-color wp-elements-5b4944fde6d3cdc5319d984d82c14527\" id=\"Purety-Verification\">Purety Verification<\/h6>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>Recommended method<\/strong>: Nanodrop or other spectrophotometric method.<\/li>\n<\/ul>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Optical density ratio<\/strong><\/th><th><strong>Expected value<\/strong><\/th><th><strong>Problems related to out-of-range values<\/strong><\/th><th><strong>Impact on sequencing<\/strong><\/th><\/tr><\/thead><tbody><tr><td>260\/280<\/td><td>Between 1.8 and 2.0<\/td><td>Contamination by proteins or phenol. Or an unstable measurement due to an insufficient concentration.<\/td><td>Poor sequencing yield or failure<\/td><\/tr><tr><td>260\/230<\/td><td>Between 2.0 and 2.2.<\/td><td>Contamination by salts, solvents, or chemical residues. Or an unstable measurement due to an insufficient concentration.<\/td><td>Poor sequencing yield or failure<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>If using a Qiagen kit<\/strong>: perform the optional PB buffer wash step to increase purity.<\/li>\n<\/ul>\n\n\n\n<div class=\"wp-block-buttons is-horizontal is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-03627597 wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button\"><a class=\"wp-block-button__link wp-element-button\" href=\"#top-of-page\">Return to top of page<\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-dbba658f65feaf71a1d85091c8e26301\" id=\"Required-containers-and-sample-identification\">Required Containers and Sample Identification<\/h4>\n\n\n\n<ul class=\"wp-block-list\">\n<li><strong>16 samples or less<\/strong>: 1.5mL tube<\/li>\n\n\n\n<li><strong>17 samples or more<\/strong>: 96-well PCR plate<\/li>\n<\/ul>\n\n\n\n<p><strong>Tube and plate identification<\/strong>:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Unique, short, and legible name.<\/li>\n\n\n\n<li>Identical to the name indicated on the submission form.<\/li>\n\n\n\n<li>Plate samples &#8211; Column arrangement required, as indicated on the form.<\/li>\n<\/ul>\n\n\n\n<p>This is to avoid any delays in processing the application.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Required tubes<\/strong> &nbsp;<\/th><th><strong>96-well PCR plate recommended<\/strong> &nbsp;<\/th><th><strong>96-well PCR plate unaccepted<\/strong> &nbsp;<\/th><\/tr><\/thead><tbody><tr><td>&#8211; 1.5mL tubes<br><br>All types of 1.5mL tubes. are accepted. &nbsp;<\/td><td>&#8211; Half-skirt PCR plate<br><br>All types of clear half-skirt clear 96-well PCR plates are accepted.<br>Example: <em>Thermo-Fast 96 PCR detection plate with flat deck; Life Technologies, Cat# AB1400L<\/em> <br><br>&#8211; Full-skirt PCR plate<br><br>All types of clear full-skirt clear 96-well PCR plates are accepted.<br>Example: <em>Microseal PCR plates 96-well clear; Bio-Rad, Cat#&nbsp;MSP9601<\/em> &nbsp;<\/td><td>&#8211; 96-well cell culture plates<br><br>&#8211; Opaque 96-well PCR plates &nbsp;<\/td><\/tr><tr><td>&nbsp;<\/td><td><strong>Plates should be sealed with 8 or 12-well strip caps or heat seals<\/strong> to ensure a tight seal of each well. <br><br>Adhesive sealants should be avoided due to the high risk of leakage and cross-contamination. &nbsp;<\/td><td>&nbsp;<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div class=\"wp-block-buttons is-horizontal is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-03627597 wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button\"><a class=\"wp-block-button__link wp-element-button\" href=\"#top-of-page\">Return to top of page<\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-1afbfc3d09342a442ee7af6d6f5edfd7\" id=\"Service-Request-Form-and-Sample-Submission\">Service Request Form and Sample Submission<\/h4>\n\n\n\n<p>Click on <a href=\"https:\/\/genomequebec.com\/en\/technological-services\/centre-dexpertise-et-de-services-2\/instructions\/service-request-and-sample-submission\/\" target=\"_blank\" data-type=\"link\" data-id=\"https:\/\/genomequebec.com\/en\/technological-services\/centre-dexpertise-et-de-services-2\/instructions\/service-request-and-sample-submission\/\" rel=\"noreferrer noopener\">service request and sample submission<\/a> to view instructions.<\/p>\n\n\n\n<p>It is <strong>highly recommended<\/strong> to provide a reference sequence for PCR product sequencing. This is to facilitate consensus sequence reconstruction if the amplicon quality is poor, especially if the sample contains more than one amplified sequence.<\/p>\n\n\n\n<p>The only accepted formats are Fasta (.fa, .fasta), SnapGene (.dna), Genbank (.gb, .gbk), Ape (.ape), text (.txt, sequence only, without header or other information).<\/p>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-096fea86e9448c1a40045488fbf1d398\" id=\"Sample-Shipment-Preparation\">Sample Shipment Preparation<\/h4>\n\n\n\n<p>Click on <a href=\"https:\/\/genomequebec.com\/en\/technological-services\/centre-dexpertise-et-de-services-2\/instructions\/sample-shipment-preparation\/\" target=\"_blank\" data-type=\"link\" data-id=\"https:\/\/genomequebec.com\/en\/technological-services\/centre-dexpertise-et-de-services-2\/instructions\/sample-shipment-preparation\/\" rel=\"noreferrer noopener\">samples shipment preparation<\/a> to view instructions.<\/p>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h4 class=\"wp-block-heading has-secondary-color has-text-color has-link-color wp-elements-c198d007b114cae3c4e0c5cfccb510db\" id=\"Transmission-of-results\">Transmission of Results<\/h4>\n\n\n\n<p>An automated message from Nanuq is sent to the submitter as soon as the sequences are available.<\/p>\n\n\n\n<p>Sequencing results are directly accessible via the <a href=\"https:\/\/ces.genomequebec.com\/nanuqAdministration\/j_security_check?service=https%3A%2F%2Fces.genomequebec.com%2FnanuqAdministration%2Fnanuq-administration%2FwelcomeHome.do\" target=\"_blank\" rel=\"noreferrer noopener\">Nanuq<\/a> web application.<\/p>\n\n\n\n<p>Each sequencing result folder contains:<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td><strong>Plasmid<\/strong><\/td><td><strong>PCR product<\/strong><\/td><\/tr><tr><td>\u2022 A consensus sequence (.fasta).<br><br>\u2022 A file with statistics for each base of the consensus sequence (.xlsx).<br><br>\u2022 Two graphs reporting the quality and size of the reads observed during sequencing (.png).<br><br>\u2022 An annotated map of the consensus sequence that can be viewed in a web browser (.html) or with software such as snapgene or snapgene-viewer (.gbk).<br><br>\u2022 An alignment of the consensus sequence with the reference sequence (if provided) (.aln). This alignment can be conveniently viewed using snapgene or snapgene-viewer software. &nbsp;<\/td><td>\u2022 A consensus sequence (.fastaq).<br><br>\u2022 A file with statistics for each base in the consensus sequence (.xlsx).<br><br>\u2022 A sequencing report that can be viewed in a web browser. &nbsp;<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p>These files can be downloaded locally.<\/p>\n\n\n\n<p>All samples will be retained for a maximum of 2 weeks after sequencing before being destroyed.<\/p>\n\n\n\n<div class=\"wp-block-buttons is-horizontal is-content-justification-center is-layout-flex wp-container-core-buttons-is-layout-03627597 wp-block-buttons-is-layout-flex\">\n<div class=\"wp-block-button\"><a class=\"wp-block-button__link wp-element-button\" href=\"#top-of-page\">Return to top of page<\/a><\/div>\n<\/div>\n\n\n\n<div style=\"height:35px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p><strong>Version 1.0_2025-10-01<\/strong><\/p>\n","protected":false},"featured_media":0,"parent":23840,"menu_order":0,"template":"","class_list":["post-32201","service","type-service","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/genomequebec.com\/en\/wp-json\/wp\/v2\/service\/32201","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/genomequebec.com\/en\/wp-json\/wp\/v2\/service"}],"about":[{"href":"https:\/\/genomequebec.com\/en\/wp-json\/wp\/v2\/types\/service"}],"up":[{"embeddable":true,"href":"https:\/\/genomequebec.com\/en\/wp-json\/wp\/v2\/service\/23840"}],"wp:attachment":[{"href":"https:\/\/genomequebec.com\/en\/wp-json\/wp\/v2\/media?parent=32201"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}