1. Enstrüman Kurulum <ol><li> Spectrofluorometer açın ve 1 saat ısınmasını bekleyin. </li><li> Excel, Şekil 1&#39;de gösterildiği gibi, Standartlar ve örnek veri ile bir dosya oluşturmak ve virgülle ayrılmış kaydetmek &quot;csv&quot; formatında değerleri. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Şekil 1" /> Şekil 1. Standartlar ve örnekler veri. </li><li> Flüorometre aşağıdaki gibi ayarlayın: <ol><li> Tıklayın <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icon 1" /> düğmesi, Şekil 2&#39;de gösterildiği Analiz Yöntemi penceresi açılacaktır. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Şekil 2" /> Şekil 2. Analiz Metodu Pencere, Genel sekmesinde. Aşağıdaki seçimleri yapın: <ul><li> &quot;Ölçüm&quot; alanına, menüyü aşağı çekme Fotometri seçin. </li><li> &quot;Operatör&quot; alanına, uygun operatörün adını girin. </li><li> &quot;Instrument&quot; alanına bilgi girmek için gerek yoktur. Bu, yazılım tarafından otomatik olarak seçilir. </li><li> Mikroplak okuyucu kullanarak, &quot;Örnekleme&quot; alanında etkin değildir. </li><li> Raporu &quot;Yorumlar&quot; alanına dahil etmek istediğiniz herhangi bir yorum girin. </li><li> &quot;Aksesuar&quot; alanına, bu test için kullanılan aksesuarlar ile ilgili bilgiler girin. </li><li> Pencerenin sağ tarafındaki düğmeleri unutmayın. </li><li> &quot;Load&quot; düğmesine önceden kaydedilmiş Analiz Yöntemleri yüklenmesini sağlar. </li><li> &quot;Kaydet&quot; düğmesine aynı adı kullanarak, test parametreleri bir dizi ya da önceden kaydedilen ve yüklenen Analiz Metodu güncellemeye izin verir. </li><li> &quot;Farklı Kaydet&quot; düğmesine test parametreleri veya Çözümleme Yöntemi, istenilen bir isim altında yeni bir dizi tasarruf sağlar. </li></ul> Şimdi &quot;kantitasyonu&quot; sekmesine tıklayın bkz. Şekil 3 ve aşağıdaki seçimleri yapın. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Şekil 3" /> Şekil 3. Analiz Metodu Pencere, kantitasyonu sekmesini. <ul><li> &quot;Kantitasyonu tipi&quot; alanında, Dalgaboyu seçin. Bu floresan okuma seçilen dalga boyu kullanılarak yapılacaktır gösterir. </li><li> &quot;Kalibrasyon&quot; alanına, 1. sırasını seçin. </li><li> &quot;Dalga boyu Numarası&quot; alanında, 1 seçin. </li><li> &quot;Konsantrasyon birim&quot; alanında, ng / ml girin. </li><li> &quot;Manuel Kalibrasyon&quot; ve &quot;sıfır yoluyla Kuvvetleri eğrisi&quot; kutuları seçilmemiş bırakın. </li><li> &quot;Rakam ondalık noktadan sonra&quot; alanında, 2 seçeneğini seçin. </li><li> &quot;Alt konsantrasyon sınırı&quot; alanına, 0 girin. </li><li> &quot;Üst konsantrasyon limiti alanında, 10000 girin. </li></ul> Şimdi &quot;Instrument&quot; sekmesine tıklayın ve aşağıdaki seçimleri yapın. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Şekil 4" /> Şekil 4. Analiz Metodu pencere, Enstrüman sekmesi <ul><li> &quot;Veri modu:&quot; alanına, Floresans seçin. </li><li> &quot;Doğrama hız&quot; alanında bu zamanda, sadece fosforesans ölçümler için kullanılan etkin değildir. </li><li> &quot;Dalga boyu modu&quot; alanında, hem WL sabit seçin. </li><li> &quot;EX / WL1&quot; alanına 480nm girin. </li><li> &quot;EM / WL1&quot; alanına 520Nm girin. </li><li> EX altında diğer tüm alanlar ve EM, şu anda devre dışıdır. </li><li> &quot;EX yarık&quot; alanına menüyü aşağı çekme 5.0nm seçin. </li><li> &quot;EM yarık&quot; alanına menüyü aşağı çekme 5.0nm seçin. </li><li> &quot;PMT Voltaj 1-1000V&quot; alanında önünde kutusu seçilmemiş bırakın. </li><li> &quot;PMT Voltage&quot; alanında, menüyü aşağı çekme 700V seçin. </li><li> &quot;Otomatik istatistik calc. Numarası&quot; alanında 2&#39;yi seçin. </li><li> 1 &quot;çoğaltır&quot; alanını seçin. </li><li> &quot;Entegrasyon&quot; alanına 0.1s seçin./ P&gt; <li> &quot;Gecikme&quot; alanına girmek veya 0s seçin. </li></ul> &quot;Monitör&quot; sekmesini tıklayın. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Şekil 5" /> Şekil 5. Analiz Metodu pencere, Monitör sekmesini. <ul><li> &quot;Y ekseni&quot;, &quot;Max:&quot; alanına, 10000 seçin </li><li> &quot;Y ekseni&quot;, &quot;Min:&quot; alanına, 0 seçin. </li><li> &quot;Açık veri işleme veri toplama sonra&quot; sol kutusunu işaretleyin. </li><li> Okumalar tamamlandıktan sonra otomatik olarak basılacak bir rapor istediğiniz sürece, &quot;veri toplama sonra Baskı raporu&quot; sol kutusu seçilmemiş bırakın. </li></ul> &quot;Rapor&quot; sekmesine tıklayın, Şekil 6 bakın. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Şekil 6" /> Şekil 6. Analiz Metodu penceresi, Rapor Tab. <ul><li> &quot;Çıkış&quot; alanına, menü aşağı çekme Rapor Yazdır seçeneğini seçin. Ayrıca ısmarlama raporlar üretmek izin Report Generator programı isteğe bağlı eklemek kullanarak eğer Excel&#39;e veri veya &quot;Kullanım Baskı Jeneratör Formu&quot; istiyorsanız, &quot;Microsoft Excel kullan&quot; seçeneğini seçin. </li><li> Rapora dahil olmasını istediğiniz öğeleri için kutuları tıklayın. </li><li> &quot;Analiz Yöntemi&quot; penceresinde farklı sekmelerde seçilen tüm parametreleri kaydetmek için, &quot;tekrar&quot; Genel &quot;sekmesini tıklayın ve bir dosya adı altında ve ileride kullanmak için onları bulmak bir yere kaydedin ve tıklayın. OK &quot;Bu pencereyi kapatmak için düğmesine basın. </li><li> Bu cihaz test parametreleri kurma tamamlar. </li><li> Şimdi mikroplak okuyucu kurar. </li><li> Tıklayın <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Simge 2" /> düğmesi, üstüne MPR Kur Penceresi getirecektir. </li></ul> MPR Kur Pencere / Plaka sekmesinde Standartlar ve Örnekler için kuyu, Şekil 7&#39;de gösterildiği gibi seçin. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Şekil 7" /> Şekil 7. MPR Kurulumu penceresinde, Levha sekmesini. <ul><li> A1 pozisyonuna E1 tıklayın ve fareyi sürükleyin, sonra üzerine tıklayın <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icon 3" /> düğmesine basın. Bu standartlar için kullanılacak pozisyonlar seçecektir. Bu pozisyonları yeşil renkte vurgulanır. </li><li> Şimdi örnekleri için H3 pozisyonları F1 seçmeniz gerekir. Sonra tıklayın tıklayın ve fareyi sürükleyin pozisyon F1 başlayarak, H3 konumuna ve <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icon 4" /> düğmesi, bu sarı renkli bu pozisyonları vurgulayacaktır. </li><li> Sonra örnek ölçümler için bu pozisyonları seçmek için, E3 A2 pozisyonlar için aynı işlemi tekrarlayın. </li><li> Şimdi, Standartlar ve örnek bilgileri önceden hazırlanmış dosya Virgülle Ayrılmış Değişken (csv) dosyası yüklemek. </li></ul> Şekil 8&#39;de gösterildiği gibi &quot;MPR Kurulum Pencere&quot;, &quot;İyi bir bilgi&quot; sekmesine tıklayın . <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Şekil 8" /> Şekil 8. MPR Kurulumu penceresinde, Kuyu Bilgi sekmesi. <ul><li> Sonra üzerine tıklayın <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Simge 5" /> düğmesine basın. &quot;Peki information.csv&quot; dosya bulma ve yükleme sağlayacaktır Şekil 9 gösterilen bir Windows Explorer penceresinde açılacaktır. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Şekil 9" /> Şekil 9. Iyi örnek bilgileri yükleniyor. &quot;Eh bilgi&quot; dosyasını yükledikten sonra, &quot;İyi bilgi&quot; Şekil 10&#39;da gösterildiği gibi görünecektir <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Şekil 10" /> Şekil 10. Örnek kuyusu bilgi yüklendi. </li></ul></li></ol></li></ol> 2. Picogreen Testi Numune Hazırlama <ol><li> 20 X TE Tampon 1 ml ekleyerek 1X TE Tamponu (10 mM Tris-HCl, 1 mM EDTA) hazırlayın19 ml di H 2 O. </li><li> 9.95 ml 1 X TE Tamponu 50 mcL 200 X PicoGreen Reaktif ekleyerek PicoGreen Reaktif Çözüm hazırlayın. </li><li> Stok dsDNA (100 mg / ml) seri dilüsyonları yaparak standartları (Lambda dsDNA) hazırlayın. İlk olarak 50 mikrogram / ml dsDNA stok dsDNA (100 mg / ml) ve 25 mcL 1 x TE Tamponu 25 mcL ekleyerek bir çözüm hazırlayın. Sonra, aşağıdaki tabloda listelenen belirtilen dsDNA hacimleri ve tampon ekleyerek standart çözümler hazırlamak: <table border="1"><tbody><tr><td> DsDNA Yoğunlaşma </td><td> Ses dsDNA </td><td> Buffer Hacmi </td></tr><tr><td> 1 mcg / mL </td><td> 50 mcg / ml dsDNA 20 mcL </td><td> 980 mcL 1 X TE Tampon </td></tr><tr><td> 0.1 mg / ml </td><td> 1 mcg / ml dsDNA 100 mcL </td><td> 900 mcL 1 X TE Tampon </td></tr><tr><td> 0.01 mcg / mL </td><td> 0.1 mg / ml dsDNA 100 mcL </td><td> 900 mcL 1 X TE Tampon </td></tr><tr><td> 0.001 mg / ml </td><td> 0.01 mcg / ml dsDNA 100 mcL </td><td> 900 mcL 1 X TE Tampon </td></tr></tbody></table></li></ol> 3. Standartlar ve örnek Floresans ölçümü <ol><li> Pipet Şekil 7 başına 150 96 plaka kuyuları A1-E1 içine her bir standart mcL, aşağıdaki tabloya göre: <table border="1"><tbody><tr><td> Iyi </td><td> Standart </td></tr><tr><td> A1 </td><td> 1 mcg / mL </td></tr><tr><td> B1 </td><td> 0.1 mg / ml </td></tr><tr><td> C1 </td><td> 0.01 mcg / mL </td></tr><tr><td> D1 </td><td> 0.001 mg / ml </td></tr><tr><td> E1 </td><td> 1 X TE Tampon </td></tr></tbody></table></li><li> Şekil 7 de gösterilmiştir H3, kuyular F1 pipetlenir 150 mcL bilinmeyen örnekleri </li><li> Çok kanallı pipet kullanmak için tasarlanmış bir çukur içine adım 1.2 &#39;de hazırlanan PicoGreen çözüm dökün. Kuyuları A1-H3 150 mcL PicoGreen çözümü sunmak için çok kanallı pipet kullanın. Hafifçe karıştırın çözüm pipet ile ve standartlara. </li><li> Plaka ve karanlık bir alanda geliştirmek için reaksiyon için 2-5 dakika bekleyin. </li></ol> 4. Temsilcisi Sonuçlar Iyi bir kalibrasyon eğrisi yazılım tarafından kalibrasyon eğrisi ve hesaplama standartları ölçümü üzerine F-7000 yazılımı tarafından sağlanan farklı parametreler kullanılarak değerlendirilir. Belirleme katsayısı, ölçülen standartlara uyum iyiliği ve hesaplanan kalibrasyon eğrisi gösterir. Yakın bu değer &quot;1&quot;, ölçülen değer ve kalibrasyon eğrisi daha iyi uyum. Değerini &quot;1&quot; uzak ise, standartlar, yeniden ölçülür tekrar hazırlanır veya kalibrasyon eğrisi fitness değişti olmalıdır. Şekil 11 PicoGreen boya kullanarak dsDNA ölçümü için elde edilen hesaplanmış bir belirleme katsayısı = 0,99993, bir kalibrasyon eğrisi bir örnek gösterir. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Şekil 11" /> Şekil 11. DsDNA kalibrasyon eğrisi örneği.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

Luis A. Moreno ve Kendra L. Cox bu yazıda kullanılan alet üreten Hitachi High Technologies Amerika tarafından istihdam edilmektedir.

Dikkatli numune hazırlama sırasında pipetleme yanı sıra, kararlı ve hassas bir floresan spektrofotometre kullanılarak iyi ve tekrarlanabilir sonuçlar elde etmek için esastır. Bu test için kullanılan floresan spektrofotometre durumda, mikroplaka okuyucu 300 mcL son numune hacmi kullanarak önerilmektedir. Düşük ses duyarlılığı azalır ve daha büyük bir hacim kuyuları arasında çapraz kontaminasyona neden olabilir.

<table><tbody><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Reagent</td><td></td><td></td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td><td>Reagent</td><td></td><td>P7589</td><td></td></tr><tr><td>Nalge Nunc Fluorescence Microplates p/n 237108</td><td>Supply</td><td></td><td>237108</td><td></td></tr><tr><td>12 Channel Eppendorf Micropipette</td><td>Supply</td><td></td><td>3516677</td><td></td></tr><tr><td>1000 &amp;mu;L Eppendorf Pipette</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>200 &amp;mu;L Eppendorf Pipette </td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>10 mL serological pipet</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Pipette tips</td><td>Supply</td><td></td><td></td><td></td></tr><tr><td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td><td>Equipment</td><td></td><td>5J1-0003</td><td></td></tr><tr><td>Microplate Reader Accessory for F-7000</td><td>Equipment</td><td></td><td>5J0-0139</td><td></td></tr></tbody></table>

quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

Niceleme özellikle küçük konsantrasyonlarda DNA, DNA sentezi ve saflaştırılması, DNA amplifikasyon ürünlerinin ölçümü gibi tanı uygulamaları ve ilaç DNA molekülleri algılama gibi standart moleküler biyoloji testleri de dahil olmak üzere biyolojik uygulamaların geniş bir yelpazede önemli bir görevdir preparatlar. Bu video sırasında dsDNA kantifikasyon Molecular Probes Quant-PicoGreen boya Reaktif kitini kullanarak gerçekleştirmek için Mikro Plaka Okuyucu aksesuar ile donatılmış F-7000 Hitachi Floresans Spektrofotometre yeteneğini gözler önüne serecektir. F-7000 Floresans Spektrofotometre, yüksek hassasiyette ve yüksek hızda ölçümler sunmaktadır. Bu ölçüm floresan, lüminesans ve fosforesans kapasitesine sahip son derece esnek bir sistem. Dalga boyu tarama, zamanlı tarama, fotometri ve 3-D tarama ölçümü de dahil olmak üzere, çeşitli ölçüm modları mevcuttur. Spektrofotometre mikroplak 300 mcL numune hacmi flöresein 50 picomoles aralığında hassasiyet vardır ve 60.000 nm / dakika tarama hızları ölçme yeteneğine sahiptir. Ayrıca konsantrasyonlarının geniş bir yelpazede üzerinde kalibrasyon eğrilerinin kullanımı için izin büyüklüğü 5 emir kadar geniş bir dinamik aralık vardır. Optik sistemi, maksimum enerji ve duyarlılık için tüm yansıtıcı optik kullanır. Standart dalga boyu aralığı 200-750 nm, ve isteğe bağlı yakın kızılötesi photomultipliers birini kullanarak 900 nm kadar uzatılabilir. Sistemi, isteğe bağlı bir dış ısı kontrollü sıvı sirkülatör kullanarak 5 ile 60 derece santigrat plaka okuyucu için isteğe bağlı sıcaklık kontrolü sağlar. Mikroplaka okuyucu kullanmak için 96 mikroplaka sağlar ve kinetik modunu kullanarak 96 kuyu için ölçüm hızı 60 saniyeden az. F-7000 ve Mikroplaka Okuyucu Yazılım kontrolleri de son derece esnektir. Örnekler sütun veya satır biçimlerinden birini ve örnek ölçümler için seçilen kuyu herhangi bir kombinasyonu olabilir. Bu optimal kullanımı için mikroplak izin verir. Ayrıca, yazılım Excel&#39;de oluşturulmuş ve virgülle ayrılmış değerler kaydedilir mikro plaka örnek konfigürasyonları, ya da &quot;csv&quot; formatında ithal sağlar. Mikroplaka ölçüm yapılandırmaları kaydedilir ve kolaylık sağlamak için yazılım ve verimlilik artışı ile geri çağrılabilir. Veri sonuçları standart bir rapor çıktı, Excel ya da isteğe bağlı Report Generator Programı olabilir.

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

DsDNA ölçümü Moleküler Problar PicoGreen boya ve bir mikroplak okuyucu aksesuar ile donatılmış Hitachi F-7000 Floresans Spektrofotometre kullanılarak gösterilmesi.

Hitachi F-7000 Floresans Spektrofotometre ve PicoGreen Boya kullanarak dsDNA Niceleme

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

quantification-dsdna-using-hitachi-f-7000-fluorescence

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Biology

24.5K Görüntüleme.  Hitachi High Technologies America. DsDNA ölçümü Moleküler Problar PicoGreen boya ve bir mikroplak okuyucu aksesuar ile donatılmış Hitachi F-7000 Floresans Spektrofotometre kullanılarak gösterilmesi.

Video: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

Eşzamanlı Niceleme için Dubleks Dijital PCR Testi<em&gt; Enterococcus spp.</em&gt; Ve Waters İnsan Fekal ilişkili HF183 Marker

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Çevre

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids gelen Viral DNA hazırlanması

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

İmmünoloji ve Enfeksiyon

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA Formazan yağış mavi ışığa maruz tarafından indüklenen esas alarak yöntemini boyama

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Biyokimya

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

Amplifikasyon tam uzunlukta HIV-1 Proviruses yakınındaki yeni nesil sıralama için

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Genetik

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Kanser Hastalarının Kan Plazma Örneklerinde Hücresiz DNA Tespiti

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Kanser Araştırmaları

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

CD4 promotörünün yakınında CAS9 kaynaklı çift iplikçik kopmasının onarımıyla ilişkili mutasyonları tanımlamak için yeni nesil dizilemenin kullanılması

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Birleştiren QD FRET Mikroakiskan Real-Time DNA Nanocomplex Self-Meclis Monitör

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biyoloji

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Nükleik Asitlerin NanoDrop Microvolume kantitasyonu

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Konsantrasyon belirlenmesi Micro hacimli Bio-spec Nano Spektrofotometre Nükleik Asitler ve Proteinler

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Dijital Nükleik Asit Kantifikasyon Tahliller Basit Toplu Saati

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...