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.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
<tr>
<td>12 Channel Eppendorf Micropipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
<tr>
<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette tips</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J1-0003</td>
<td>&nbsp;</td>
<tr>
<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J0-0139</td>
<td>&nbsp;</td>		</tbody>
		</table>
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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

Research

JoVE Journal

Biology

24.4K 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

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

NanoDrop 2000c Spektrofotometre kullanılarak Microvolume Protein Konsantrasyonu Tayini

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Biyoloji

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

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 ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Toz numuneler mutlak Kuantum Verimi Ölçümü

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Görselleştirme Caenorhabditis Elegans Tırnak Yapıları Lipofilik Vital Boya, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Gözlem ve telomer Niceleme ve Tekrarlayan Diziler kullanma Floresan<em&gt; In Situ</emPNA Sondalar&gt; ile hibridizasyon (FISH)<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

[18F]FDG Micro-PET/MR Görüntüleme Kullanılarak Farelerde Kahverengi ve Bej Yağ Dokularının Görselleştirilmesi ve Ölçülmesi

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Tek Hücreli Floresan Görüntüleme Kullanılarak Interbakteriyel Rekabetin Nicelemesi

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...