{"id":26048,"date":"2022-02-21T18:18:07","date_gmt":"2022-02-21T10:18:07","guid":{"rendered":"https:\/\/www.accscicn.com\/?p=26048"},"modified":"2022-10-20T17:51:08","modified_gmt":"2022-10-20T09:51:08","slug":"app-mantis-2022022103","status":"publish","type":"post","link":"https:\/\/www.accscicn.com\/en\/insight\/application\/app-mantis-2022022103\/","title":{"rendered":"Fast, accurate and flexible DNA quantification using the QuantiFluor\u00ae dsDNA System on the MANTIS\u00ae Liquid Processor"},"content":{"rendered":"<div data-elementor-type=\"wp-post\" data-elementor-id=\"26048\" class=\"elementor elementor-26048\" data-elementor-post-type=\"post\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-6cc1b2cb elementor-section-full_width elementor-section-stretched elementor-section-height-default elementor-section-height-default\" data-id=\"6cc1b2cb\" data-element_type=\"section\" data-settings=\"{&quot;stretch_section&quot;:&quot;section-stretched&quot;,&quot;background_background&quot;:&quot;classic&quot;}\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-top-column elementor-element elementor-element-17adffd4\" data-id=\"17adffd4\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-5a039472 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"5a039472\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-609d0d23\" data-id=\"609d0d23\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-72410ec1 elementor-widget elementor-widget-heading\" data-id=\"72410ec1\" data-element_type=\"widget\" data-widget_type=\"heading.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t<style>\/*! elementor - v3.21.0 - 20-05-2024 *\/\n.elementor-heading-title{padding:0;margin:0;line-height:1}.elementor-widget-heading .elementor-heading-title[class*=elementor-size-]>a{color:inherit;font-size:inherit;line-height:inherit}.elementor-widget-heading .elementor-heading-title.elementor-size-small{font-size:15px}.elementor-widget-heading .elementor-heading-title.elementor-size-medium{font-size:19px}.elementor-widget-heading .elementor-heading-title.elementor-size-large{font-size:29px}.elementor-widget-heading .elementor-heading-title.elementor-size-xl{font-size:39px}.elementor-widget-heading .elementor-heading-title.elementor-size-xxl{font-size:59px}<\/style><h3 class=\"elementor-heading-title elementor-size-default\">Fast, accurate and flexible DNA quantification using the QuantiFluor\u00ae dsDNA System on the MANTIS\u00ae Liquid Processor\n<\/h3>\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<div class=\"elementor-element elementor-element-58cf909 elementor-widget elementor-widget-text-editor\" data-id=\"58cf909\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t<style>\/*! elementor - v3.21.0 - 20-05-2024 *\/\n.elementor-widget-text-editor.elementor-drop-cap-view-stacked .elementor-drop-cap{background-color:#69727d;color:#fff}.elementor-widget-text-editor.elementor-drop-cap-view-framed .elementor-drop-cap{color:#69727d;border:3px solid;background-color:transparent}.elementor-widget-text-editor:not(.elementor-drop-cap-view-default) .elementor-drop-cap{margin-top:8px}.elementor-widget-text-editor:not(.elementor-drop-cap-view-default) .elementor-drop-cap-letter{width:1em;height:1em}.elementor-widget-text-editor .elementor-drop-cap{float:left;text-align:center;line-height:1;font-size:50px}.elementor-widget-text-editor .elementor-drop-cap-letter{display:inline-block}<\/style>\t\t\t\t<p><strong>Introduction<\/strong><\/p><p>Nucleic acid quantification is an important step in molecular biology workflows, especially for advanced applications such as qPCR and next-generation sequencing (NGS). In NGS, the ultimate goal is to generate highly complex libraries and sequence them with maximum depth and homogeneity, so accurate quantification is particularly important. Two key steps to accurately determine nucleic acid concentration are:<\/p><ol><li>Maximum enzyme efficiency is sensitive to the amount of DNA input prior to library preparation and 2) after library preparation to standardize samples for pooling prior to sequencing. In addition, the need for increasingly smaller sample inputs (e.g., for RNA-seq experiments) and the challenge of isolating nucleic acids from many common sample types (e.g., FFPE and ccfDNA) make concentration estimation difficult.<\/li><\/ol><p>While multiple quantification methods exist, fluorescent dye-based systems are recommended for advanced downstream applications due to their sensitivity, target selectivity, cost, and ease of use. With the QuantiFluor\u00ae dsDNA System, a stock solution of fluorescent dye is diluted to form a working solution, which is added to a microtiter plate containing a small volume of purified DNA sample, and fluorescence is measured using a fluorometer. The sensitive and accurate QuantiFluor\u00ae dsDNA System can be used to quantify dsDNA in single tubes as well as 96- and 384-well plates.<\/p><p>Ensuring accuracy during repetitive pipetting of small volumes can be difficult and the process is labor intensive, with errors and costly rework. As a result, most laboratories requiring higher throughput are opting for automated robotic liquid handling platforms for quantitative analysis. the MANTIS\u00ae is a new benchtop liquid handler that meets many of the requirements for high-throughput reagent dispensing at a fraction of the cost of larger systems. For nucleic acid quantitation, the MANTIS\u00ae accurately dispenses small volumes of QuantiFluor\u00ae dye working solution for reproducible quantitation results. In this application note, we demonstrate the compatibility of the MANTIS\u00ae Liquid Processor to quickly and accurately automate reagent processing for QuantiFluor\u00ae dsDNA systems in both 96-well and 384-well plate formats.<\/p><p>\u00a0<\/p><p><strong>Required materials<\/strong><\/p><p style=\"padding-left: 40px;\">- MANTIS\u00ae Instrument (Formulatrix)<\/p><p style=\"padding-left: 40px;\">- High Capacity Chip (Formulatrix Cat.# MCHS6)<\/p><p style=\"padding-left: 40px;\">- QuantiFluor\u00ae dsDNA System (Cat.# E2670)<\/p><p style=\"padding-left: 40px;\">- Nuclease-free water (Cat.# P1195)<\/p><p style=\"padding-left: 40px;\">- Black flat-bottomed 96-well plate (Corning Cat.# 3915) or 384-well plate (Corning Cat.# 3573)<\/p><p style=\"padding-left: 40px;\">- 15ml or 50ml conical tubes<\/p><p style=\"padding-left: 40px;\">- Multi-well detector for fluorescence measurement (e.g., GloMax Discover System [Cat.# GM3000])<\/p><p>\u00a0 \u00a0<\/p><p><strong>Experimental program<\/strong><\/p><p>Reagent Preparation.\u00a0<\/p><ol><li><p>Prepare 1X TE Buffer: Dilute 20X TE Buffer 20 times with nuclease-free water.<\/p><\/li><li><p>Prepare a working solution of QuantiFluor\u00ae dsDNA dye.<\/p><p>a. 96-well format: Dilute QuantiFluor\u00ae dsDNA dye 1:400 in 1X TE buffer. Refer to QuantiFluor\u00ae dsDNA System Technical Manual #TM346 for detailed instructions on use.<\/p><p>b. 384-well format: Dye dilutions are based on your desired analytical requirements. (See Application Note for High-Throughput, Low-Volume DNA Quantification with the QuantiFluor\u00ae dsDNA System for information on how to select the appropriate dye dilution. ) Here we use a 1:200 dilution of QuantiFluor\u00ae dsDNA dye in 1X TE buffer to quantify 0.05-50ng DNA.<\/p><\/li><li><p>Preparation of Standard Curve: Serial dilutions were made using a known concentration of dsDNA standard and 1X TE buffer that extends up and down the concentration range of any unknown samples. For blank samples, use 1X TE Buffer.<\/p><\/li><\/ol><p>Instrument settings.<\/p><ol><li><p>Open the MANTIS\u00ae desktop application and select File &gt; New Distribution List. Select your determination plate from the list.<br \/><img fetchpriority=\"high\" decoding=\"async\" class=\"size-large wp-image-26050 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/4-1024x551.png\" alt=\"\" width=\"1024\" height=\"551\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/4-1024x551.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/4-300x161.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/4-768x413.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/4.png 1242w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><\/li><li><p>The software will prompt the user to add the reagent to the distribution list. Enter \"QuantiFluor dsDNA\" in the \"Reagent Name\" field and select \"OK\" when finished.<br \/><img decoding=\"async\" class=\"size-large wp-image-26051 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/5-1024x549.png\" alt=\"\" width=\"1024\" height=\"549\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/5-1024x549.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/5-300x161.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/5-768x412.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/5.png 1242w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><\/li><li><p>Assign QuantiFluor dsDNA to microarray position #1<br \/><img decoding=\"async\" class=\"size-large wp-image-26052 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/6-1024x549.png\" alt=\"\" width=\"1024\" height=\"549\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/6-1024x549.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/6-300x161.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/6-768x412.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/6.png 1242w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><\/li><li><p>Place the QuantiFluor\u00ae dsDNA Working Solution in the reagent rack at microarray position #1. Place the tube from the chip into the reagent.<\/p><\/li><li><p>Fill the chip and set up a new liquid dispense by selecting the desired hole and entering the target volume for the selected hole<\/p><p>a. 96-well format: Dispense 200 \u00b5l of working solution into each well.<\/p><p>b. 384-well format: dispense 36 \u00b5l of working solution into each well.<br \/><img loading=\"lazy\" decoding=\"async\" class=\"size-large wp-image-26053 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/7-1024x548.png\" alt=\"\" width=\"1024\" height=\"548\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/7-1024x548.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/7-300x161.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/7-768x411.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/7.png 1244w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><\/li><li><p>Add the microplate to the MANTIS\u00aedeck and press Start.<\/p><\/li><\/ol><p>\u00a0<\/p><p>Analytical Determination:<\/p><ol><li><p>Manually pipette standards, blanks and unknown samples into their respective wells of the multiwell plate.<\/p><p>c. 96-well format: add 1-20 \u00b5l of standards, blanks, and unknown samples.<\/p><p>d. 384-well format: add 4\u00b5l of standard, blank and unknown.<\/p><\/li><li><p>Mix the microplate thoroughly by pipetting the liquid in each well using a plate shaker or by pipetting. Failure to mix thoroughly will result in variations in readings between wells. Also avoid introducing air bubbles, which can interfere with fluorescence.<\/p><\/li><li><p>Incubate the assay for 5 minutes at room temperature.<\/p><\/li><li><p>Detects fluorescence (504nmEx\/531nmEm). If using a GloMax\u00ae Systems detection instrument, select the preloaded protocol: \"QuantiFluor dsDNA System\".<\/p><\/li><li><p>Calculate the dsDNA concentration as follows: remove the fluorescence of the blank sample (1X TE buffer) from each standard and sample fluorescence. Generate a standard curve of fluorescence versus DNA concentration using calibration data from the DNA standards. Determine the DNA concentration of any unknown samples in the standard curve by linear or power regression.<\/p><\/li><\/ol><p>Standardization.<\/p><ol><li>Concentration values from the GloMax\u00ae detection instrument can be imported directly into the MANTIS\u00ae software to calculate the volume required for standardization (see Figures 1 and 2).<\/li><\/ol><p><img loading=\"lazy\" decoding=\"async\" class=\"size-large wp-image-26054 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/8-1024x306.png\" alt=\"\" width=\"1024\" height=\"306\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/8-1024x306.png 1024w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/8-300x90.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/8-768x229.png 768w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/8.png 1038w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/p><p>Figure 1. Quantification of the K562 Human Genomic DNA Standard (Cat.# E4931) using the MANTIS\u00ae Liquid Processor to dispense QuantiFluor\u00ae dsDNA Dye working solutions in 96-well plates (Figure A) and 384-well plates (Figure B). Fluorescence was measured on the GloMax\u00ae Discover System and unknown samples were inserted from within the power regression standard curve.<\/p><p><img loading=\"lazy\" decoding=\"async\" class=\"size-large wp-image-26055 alignnone\" src=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/9.png\" alt=\"\" width=\"1016\" height=\"320\" srcset=\"https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/9.png 1016w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/9-300x94.png 300w, https:\/\/www.accscicn.com\/wp-content\/uploads\/2022\/02\/9-768x242.png 768w\" sizes=\"(max-width: 1016px) 100vw, 1016px\" \/><\/p><p>Figure 2. Quantification of the 2,200bp PCR product used to model the average size of mRNA transcripts. The Mantis Liquid Processor was used to dispense the QuantiFluor\u00ae dsDNA System in 96-well plate format (Figure A) and 384-well plate format (Figure B). Fluorescence was measured on the GloMax\u00ae Discover System and unknown samples were inserted from within the power regression standard curve.<\/p><p>\u00a0<\/p><p><strong>Summary<\/strong><\/p><p>The MANTIS\u00ae Liquid Processor provides researchers with a flexible workstation to build a variety of experimental protocols in minutes. Execute these protocols precisely to improve laboratory efficiency and experimental reproducibility. This application note demonstrates the ease of use of liquid handling software, which allows for a quick and seamless transition to an automated platform. Using the MANTIS\u00ae Liquid Handler to add reagents to the QuantiFluor\u00ae dsDNA System enables reproducible quantitation in multilayer microtiter plates with little to no instrument programming.<\/p><p>\u00a0<\/p><p>Source: @BostonFummerle<\/p><p>Time: 2022.02.15<\/p>\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-1bbf8b72\" data-id=\"1bbf8b72\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-4fe93b6c elementor-widget-divider--view-line elementor-widget elementor-widget-divider\" data-id=\"4fe93b6c\" data-element_type=\"widget\" data-widget_type=\"divider.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t<style>\/*! elementor - v3.21.0 - 20-05-2024 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\"\t\t<\/a>\n\n\t\t\t\t<\/div>\n\t\t\t\t<\/article>\n\t\t\t\t<\/div>\n\t\t\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<section class=\"elementor-section elementor-inner-section elementor-element elementor-element-429d0882 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"429d0882\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-7767bcce\" data-id=\"7767bcce\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap\">\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t<div class=\"elementor-column elementor-col-50 elementor-inner-column elementor-element elementor-element-6cca9e69\" data-id=\"6cca9e69\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap\">\n\t\t\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>","protected":false},"excerpt":{"rendered":"<p>\u5728 MANTIS\u00ae \u6db2\u4f53\u5904\u7406\u5668\u4e0a\u4f7f\u7528 Q [&hellip;]<\/p>","protected":false},"author":1,"featured_media":26059,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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