Laboratuarımızda NIGMS-Ulusal Sağlık Enstitüsü, Ulusal Bilim Vakfı ve ALANI hibe R15GM86839 KARİYER ödül MCB0551798 tarafından desteklenmektedir.

1. Büyüme ve UV Işınlama. <ol><li> Şekeri% 0.4,% 0.2 casamino asitler, ve 10 mg / ml timin (DGCthy orta) ve 100 mg / ml ampisilin ile desteklenmiş Davis orta 1 yetiştirilen plazmid pBR322 içeren taze bir gece kültür 200μl pelet. Hücre pelletini sonra ampisilin eksik 200μl DGCthy orta yeniden bekletildi ve 20 ml DGCthy orta aşılamak için kullanılır. </li><li> Kültürler 0.5 OD 600 (~ 5 x 10 8 hücre / ml) 37 sallayarak inkübatör ampisilin seçimi ° C olmadan yetiştirilmektedir. Büyüme ampisilin olmadan bazı mutantlar ortaya çıkabilecek anormal veya verimsiz çoğaltma ara karşı seçimi önler. Hasara neden UV ışık kullanıyorsanız, UV etkili doz azaltılması, bu dalga boylarında ve kalkanlar hücreleri güçlü emer çünkü ek olarak, medya ampisilin çıkarılması gereklidir. </li><li> Sarı ışıklar altında çalışan, kültür, ajitasyon için dönen bir platform üzerinde 15 cm çaplı Petri kabı yerleştirilir. Bizim kültür UVC fotometre ile ölçülür ~ 1 J/m2/sec, bir maruz kalma oranı üreten bir antiseptik lamba 15 watt bir mesafede yerleştirilir. Kültürler 50 J/m2 ışınlanmış ve ardından hemen arkasına yerleştirilen deney süresi boyunca 37 ° C inkübatör sallayarak. Bu doz, ortalama 1 Siklobütanın pirimidin dimer ssDNA her 4,5 kb, üretir. Sarı ışık fotolizaz Siklobütanın pirimidin dimerleri photoreactivation-ters önler. </li></ol> 2. DNA İzolasyonu. <ol><li> Çoğaltma ara incelenmesi gereken zamanlarda, kültür 0.75 ml kısım 0.75 ml buz gibi NET30 Buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 30 mM EDTA) içine yerleştirilir ve sonuna kadar buz üzerinde yerleştirilen zaman elbette. Genellikle 0 ile incelenen örneklerin, 15, 30, 45, 60 ve 90 dakika ile 90 dakikalık bir zaman ders çalıştırmak. EDTA ve soğuk etkili çoğaltma ve nükleotid eksizyon onarımı durdurmak için hizmet vermektedir. </li><li> Her bir örnek, 1.5 mg / ml lizozim ve TE 0.2 mg / ml RNaseA (10 mM Tris, pH 8.0, 1 mM EDTA) 150 ul yeniden süspanse ve 20 dakika 37 ° C parçalanmış, sonra pelet. Şu anda, 20% sarkosyl proteinaz K (10mg/ml) ve 10μl 10μl eklenir ve inkübasyon 1 saat süreyle devam etmesine izin. Proteinaz K ve sarkosyl fenol ekstraksiyonu oluşmadan önce veya membran proteinleri ile ilişkili olabilir DNA parçalarının serbest bırakmak için yardımcı olur. Aktif kopyalayan DNA genellikle protein veya membran komplekslere bağlı olduğundan, bu kurtarıldı çoğaltma parçaları verimi artırmaya yardımcı olur. </li><li> Örnekler daha sonra her bir örnek için fenol 2 hacim ekleme ve tüpler 5 dakika hafifçe ters tarafından ayıklanır. Sonra 2 hacim kloroform / izoamil alkol (24 / 1) eklenir ve tüpler 5 dakika boyunca yavaşça tekrar ters. </li><li> Örnekleri, 5 dakika ve her numunenin üst sulu faz için bir mikrosantrifüj 14.000 rpm&#39;de santrifüj kaldırılır ve yeni bir tüp yerleştirilir. Sonra kloroform / izoamil alkol (24 / 1) 4 cilt, eklenir tüpler 5 dakika hafifçe ters ve yine 5 dakika 14.000 rpm&#39;de santrifüj. </li><li> Her numune üst, sulu faz sonra 1 saat süreyle bir beher 0.1x TE 250 ml üzerinde yüzen bir 47mm whatman 0.025 mikron gözenek diske her numunenin 100μl lekelenme diyalize. Tek iplikli bölgeler veya şube noktaları ile kopyalayan yapılar kesme daha duyarlı olduğundan, biz genellikle ağız geniş ve kesme güçleri en aza indirmek için bir jilet ile pipet uçları kesilir. Genel olarak, pipetleme DNA restriksiyon enzimleri ile sindirilir edildikten sonra kadar minimumda tutulması olmalıdır. </li><li> Her bir örnek daha sonra çoğaltma kökeni sadece mansap plazmid linearizes PvuII (New England Biolabs) ile sindirilir. </li><li> Agaroz jel, 100μl kloroform ve Bromophenol Mavi ve Ksilen Cyanol içeren 6X yükleme boya 20μl yüklemeden önce her bir örnek eklenir ve karıştırılır. Daha sonra, yükleme boya içeren sulu faz 40μl jel içine yüklenir. Yapısal önyargıları, yapısal eserler, ve DNA kesme en aza indirmek için, bu izolasyon prosedürü hücre parçalama tek iplikli bölgelerde, çökelti, ya da DNA örnekleri konsantre zenginleştirmek için herhangi bir adım içermez. </li></ol> 3. 2D Jel ve Güney Analizi. <ol><li> 2-boyutlu agaroz-jel analizi 3 ila güncellenmiştir. 1. boyut için kısıtlı DNA örnekleri 1V/cm 1X TBE% 0.4 agaroz jel ile çalıştırılır. 10X TBE stok solüsyonu Bir Litre içerir: <ul><li> 108 g Tris Base </li><li> 55 gr Borik Asit </li><li> 40 ml 0,5 M EDTA (pH 8.0) </li></ul></li><li> Lambda Hind III boyut işareti ilk şeritte yüklenir, daha sonra örnekleri diğer her kulvarda yüklenir. Biz genellikle ilk d çalıştırmakimension ~ 12-15 saat. Şeritleri Atlama, daha kolay şerit ikinci boyutta döküm dilim hale getirir. Düşük voltaj ve düşük yüzde agaroz jel öncelikle boyutu dayalı DNA parçalarının ayrı hizmet vermektedir. </li><li> Ikinci boyut için büyük bir kasap bıçağı ile ilk boyut jel, jel şeritleri dışında dilimlenmiş. Lambda Hind III işaretleyici içeren ilk şeritli ethidium bromid ile boyandı. Bir kılavuz, ürün olarak lambda Hind III kalem kullanarak ve Lineerleştirilmiş plazmid sürmesi bekleniyor nerede aşağıda jel bölgedeki atın. Bu koşullar altında, biz bu lineerleştirilmiş pBR322 biraz ksilen cyanol leke yukarıdaki çalışır. </li><li> Ikinci boyut döküm için, her şeritli boş bir jel tekerin üstüne yatay olarak yerleştirilir. 1X TBE% 1.0 agaroz çözeltisi 55 ° C&#39;ye kadar hazırlanır ve soğutulur Şu anda, jel solüsyon, jel dilim tamamen karşılamak için emin olun, ikinci boyut döküm dökülür. Sonra jel seti, tampon sirküle sağlayan bir elektroforez biriminde 6.5V/cm çalıştırmak. Biz genellikle ikinci boyut ~ 5,5-7 saat çalıştırın. Yüksek gerilim ve yüksek yüzde agaroz jel şekline göre DNA parçalarının yanı sıra, boyutu etkin bir şekilde ayırır. Doğrusal olmayan şekiller ikinci boyut daha yavaş çalışır. </li><li> Ardından elektroforez, jel sonra H 2 O durulanır, H 2 O ile durulanır 15 dakika, 0.25m hidroklorik asit 400ml iki kere yıkanır, ve sonra iki kez 30 dakika süreyle 400 ml 0.4M NaOH yıkanır . Asit DNA molekülleri hizmet daha verimli bir şekilde aktarma küçük parçalara kısmen nick yıkar. Bu adım, büyüklüğü ve DNA replikasyonu ara alışılmadık şekillere nedeniyle gereklidir. </li><li> Jeller DNA sonra bir Hybond N + naylon membran 4. Biz 0.4M NaOH ile alkali aşağı yönlü bir transfer sistemi kullanır. Kısaca, iki sayfa 0.4M NaOH batırılmış kağıt blot büyük bir yığın kağıt havlu üzerine yerleştirilir. Naylon membran 0.4M NaOH batırılmış ve blot kağıt üstüne yerleştirilir. Sonra jel dikkatle NaOH çözeltisi ıslatılmış başka bir parça kurutma kağıdı takip Bunun üstüne katmanlı. Son olarak, uzun bir parça ıslak blot kağıt üst kısmında katmanlı ve iki ucu, 1 L bir fitil olarak hizmet 0.4M NaOH çözeltisi içeren bir yemekleri yerleştirilir. Biz genellikle 6-12 saat için DNA transferi sağlar. </li><li> Naylon zarı kaldırılır, 5X SSC Tampon 20-30 sn yıkanmış ve 10.5ml Prehybridization Çözüm içeren bir melezleşme silindir şişede yerleştirilir. Sonra, membran 42 ° C rotasyon ile fazla 6 saat inkübe edilir. Prehybridization çözüm: <ul><li> 5.0 ml formamid </li><li> 0.5 ml% 20 SDS </li><li> 2.5 ml 20X SSC * </li><li> 2.0 ml 50X Denhardt * </li><li> 0.5 ml Somon Sperm DNA (10mg/ml) * SSC ve Denhardt için yemek tarifleri 5 bulunabilir </li></ul></li><li> Plazmid pBR322 prehybridization dönemde, 1 mg [32-P]-dCTP etiketli kullanarak alfa Roche tarafından verilen protokole göre nick çeviri 32P ile etiketlenir. Radyoaktif prob (100μl), 98 inkübe tarafından denatüre ° C, daha sonra bir vidalı kapak mikrosantrifüj tüp içinde 10 dakika boyunca ve buz üzerinde hemen önüne yerleştirilmelidir. </li><li> Denatüre prob içeren 5.9 ml hibridizasyon çözüm eklenir: <ul><li> 3.0 ml formamid </li><li> 1.5 ml SSC </li><li> 1.0 ml H 2 O </li><li> 0.15 ml 50X Denhardt </li><li> 0.10 ml% 20 SDS </li><li> 0.15 ml 10mg/ml Somon Sperm DNA </li></ul></li><li> Prehybridization çözüm silindir şişe dışarı dökülür ve hibridizasyon çözümü silindir şişe sonra 12hrs daha fazla 42 ° C inkübatör ve döndürülmüş iade edilir dökülür. </li><li> Hibridizasyon çözüm silindir şişe dışarı dökülür. Sonra, leke silindir rotasyon ile 0.5x SSC,% 0.1 SDS 42 ° C yıkama solüsyonu içeren bir ~ 150 ml şişe içinde 20 dakika 4 kez yıkanır. </li><li> Kadar sıvı gözle görülür bir şekilde ortadan kayboldu son yıkama sonrasında, membran bir kağıt havlu üzerine yerleştirilir. Membran sonra polivinil klorür plastik şal sarılmış ve bir phosphorimager ekrana maruz kalmaktadır. Fırtına 840 ve ilişkili ImageQuant Yazılım kullanarak radyoaktivite görselleştirmek ve kantitatif. </li></ol> 4. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="Şekil 1" /> Şekil 1. Plazmid çoğaltma ara UV ışınlarına bağlı DNA hasarı varlığı ve yokluğu gözlenen PvuII göç desen 2D-agaroz-jel analizi ile gözlenen plazmid pBR322 sindirilir diyagramı. Sigara kopyalayan doğrusal plazmid 4.4-kb doğrusal bir parçası olarak çalıştırın. Çoğaltılıyor plazmid, onların daha büyük boyutuna bağlı olmayan kopyalayan parçaları daha yavaş ve göç Y-şekilli yapılar formunlinear şekli. Bu göç desen doğrusal bölgede iyi doğru uzanan bir yay oluşturur. UV ışınlama, çift-Y ya da X-biçimli molekülleri, yay Y-şekilli moleküllerin daha yavaş göç koni bölgede görülmektedir. 2D jel Güney analizi bir örnek 32P-etiketli pBR322 probed UV ışını ve 15 dakika UV ışını (Yayıncının izni ile çoğaltılabilir) sonra hemen sonra hücreler için gösterilmiştir.

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

UV hasarına varlığı ve yokluğu vahşi tip hücreler elde edilen tipik sonuçlar Şekil 1&#39;de gösterilmiştir. Hasar yokluğunda, hücreler üstel fazda hızla büyüyor ~% 1, toplam plazmid DNA Y ark. Bloke çoğaltma çatal hasarlı siteleri biriktikçe ışınlama takiben, Y şekilli moleküllerin geçici bir artış görülmektedir. X-şeklinde çoğaltma ara lezyonlar tamir ile ilişkilendiren bir saatine kadar geçici olarak birikir ve devam. Güney analizi yerine, çoğaltma a...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
</tr>
<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
</tr>
<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
</tr>
<tr>
<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
</tr>
<tr>
<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
</tr>
<tr>
<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
</tr>
<tr>
<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
</tr>		</tbody>
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visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

DNA hasarı varlığı Yanlış çoğaltma ve yaygın olarak insanlarda kanser gelişimi ile doğrudan ilişkili olduğuna inanılan tüm hücre tiplerinde gözlenen hücresel düzenlenmeleri ve mutagenez çoğunluk için sorumludur. DNA hasarı, UV ışını ile indüklenen gibi ciddi doğru genomik şablonu çoğaltmak çoğaltma yeteneğini bozar. Bir dizi gen ürünleri çoğaltma şablon DNA lezyonlar karşılaştığında gerekli olduğu tespit edilmiştir. Ancak, kalan meydan çoğaltma sırasında bu proteinlerin nasıl süreci lezyonları belirlemek olmuştur<em&gt; In vivo</em&gt;. Escherichia coli bir model sistem olarak kullanarak, iki boyutlu agaroz-jel analizi kopyalayan plazmid ortaya çıkan yapısal ara tanımlamak için kullanılabilecek bir prosedür tarif<em&gt; In vivo</em&gt; UV ışınlarına bağlı DNA hasarını. Bu prosedür, UV hasarına tarafından bloke çoğaltma çatal, reca ve RecF yolağı ile ilişkili çeşitli gen ürünleri tarafından stabilize geçici bir ters tabi olduğunu göstermek için kullanılmıştır. Bu teknik, bu çoğaltma ara nükleotid eksizyon onarımı ve çoğaltma devam eder lezyonların kaldırılması ile ilişkilendiren bir saatine kadar korunur olduğunu gösteriyor.

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Biz, iki boyutlu agaroz-jel analizi, UV ışını meydana çoğaltma ara yapısını tanımlamak için kullanılır olabilir bir prosedür sunuyoruz.

UV ile indüklenen Çoğaltma Ara Görselleştirme E. coli İki boyutlu agaroz-jel Analizi

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

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Biology

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

10.6K Görüntüleme.  Portland State University. Biz, iki boyutlu agaroz-jel analizi, UV ışını meydana çoğaltma ara yapısını tanımlamak için kullanılır olabilir bir prosedür sunuyoruz.

Video: Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Isı Şoku Yöntemiyle E. coli içine Plazmid DNA Dönüşüm

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Biyoloji

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Nöral Kök Hücre Motilite değerlendirilmesi Agaroz Jel tabanlı mikroakışkan Cihaz Kullanımı

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Yarı denatüre Deterjan-Agaroz Jel Elektroforez Amiloid Toplama taraması

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Proteinlerin Yönetmen Evolution Mutagenez ve Fonksiyonel Seçim Protokolleri E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

DNA replikasyonu Görselleştirme Omurgalı Model Sistemi DT40 DNA Fiber Tekniği

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

DNA kırıntıları Ayrılma Agaroz Jel Elektroforezi

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

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 ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Hedeflenen Srna faj aracılı teslim Gen İfade Knock Down için oluşturur<em&gt; E. E. coli</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

İnsan Norovirüs çıkıntılı Domains üretimi içinde<em&gt; E. E. coli</em&gt;, X ışını Kristallografi için

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Musin Agaroz jel elektroforezi: Yüksek molekül ağırlıklı glikoproteinler için Western Blot


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...