1. Lizat Gümrükleme <ol><li> E. büyütün Luria Broth 37 gecede uygun antibiyotik ile derin kuyu plakaları istenen plazmid DNA (kültür / iyi 1 ml) ° C sallayarak ile dönüştürdü coli kültürleri. </li><li> 10 dakika boyunca 5.000 xg&#39;de kültür plakası Pelet E. coli, sonra süzün süpernatant. </li><li> 100 mcL Tekrar Süspansiyonu Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mikrogram / mL RNaz A) her pelletini tekrar. </li><li> 100 mcL / iyi Lizis Buffer (200 mM NaOH,% 1 SDS) ekleyin ve 2 dakika boyunca plaka çalkalayıcı kullanarak sallamak. </li><li> 100 kuyu / mcL Nötralizasyon Buffer (3.0 M potasyum asetat, pH 5.5) ekleyin ve 2 dakika çalkalanır. </li><li> Açıklama plaka lizat hücre lizat aktarın. </li><li> 350 mcL toplama plakası ve vakum manifoldunun altına yeri üstüne yerleştirin DNA bağlayıcı plaka. (Bkz. Şekil 1) vakum manifoldunun birleştirin. </li><li> Elektrikli cihazların üstüne yerleştirin lizat açıklama plakası. </li><li> 10 inç Hg (0,34 bar) vakum uygulayın ve DNA bağlayıcı plakasına süzüntü toplamak. Hg in 12 daha büyük vakum kullanmayın. (0.41 bar). </li><li> Vakum manifoldunun sökün. Aparat altına yerleştirin 2 mL atık toplama plakası. </li></ol> 2. DNA Bağlama <ol><li> (Bkz. Şekil 2) DNA bağlayıcı plaka manifoldunun üstüne taşıyın. </li><li> 300 mcL / iyi Bağlama Buffer (6 M Guanidin-HCl) ekleyin ve karıştırın ve aşağı yukarı pipetle. </li><li> Vakum uygulayın [~ Hg yavaş vakum inç 5 (0.17 çubuğu)] ve süzüntü atın. DNA membrana bağlı. </li><li> Yıkama Tamponu (80% Etanol) / iyi 400 mcL ile yıkayınız. </li><li> Vakum uygulayın, sonra süzüntü atın. 2 mL toplama plakası kullanılarak veya doğrudan atık filtreleme, gereksiz yere atın. </li><li> Tekrar bir kez yıkayın. </li><li> Leke alt kurumaya havluya filtre plaka. </li><li> Artık alkol kaldırılmasını sağlamak için 5-10 dakika bir kez daha vakum uygulayın. </li><li> Etanol damlacıkların kaldırılmasını sağlamak için alt kurutun. </li></ol> 3. DNA Elüsyon <ol><li> 70 kuyu / mcL Elüsyon Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) ekleyin. Yüksek DNA konsantrasyonu isteniyorsa, hacim 50 mcL / iyi olabilir, ama ortalama toplam verim düşük olacaktır. </li><li> Plaka, bir dakika oda sıcaklığında inkübe edin. </li><li> Arıtılmış DNA ya vakum veya santrifüj tarafından yıkandı, olabilir. </li><li> Vakum yöntemi: Vakum manifoldu içine yerleştirin temiz toplama plakası (DNaz, RNaz ücretsiz). Tüm elüsyon tampon DNA bağlayıcı plaka geçti kadar vakum manifoldunun üstüne koyun filtre plakası, 15 inç Hg (0,5 bar) 1 dakika için vakum uygulamak. Saflaştırılmış DNA (bkz. Şekil 3) toplayın. </li><li> Santrifüj yöntemi: 5 dakika 1000 xg&#39;de temiz toplama plakası ve santrifüj üst üste yerleştirin arıtma filtre plakası. </li></ol> 4. Nicel ve Nitel Analiz <ol><li> OD 260/280 ölçün, saflaştırılmış DNA konsantrasyonu hesaplar. </li><li> Saflaştırılmış pDNA agaroz jel analizi gerçekleştirin. </li></ol> 5. Temsilcisi Sonuçlar E. coli, malzeme başlayan enkaz kayda değer bir azalma gösterdi geleneksel santrifüj ve Pall AcroPrep Advance lizat açıklama Filtre Tabaklar kullanarak açıklık lizat . (Şekil 4). Üç konsantrasyonlarda Plazmid DNA TE tampon içine çivili ve AcroPrep Advance lizat açıklama filtre plakası üzerinden geçti. Plazmid DNA konsantrasyonunun karşılaştırılması, filtrasyon öncesi ve sonrası, gösteriler üç konsantrasyonlarda% 100 iyileşme (Şekil 5) ~. Açıklık E. DNA kurtarma göstermek için coli lizat, plazmid DNA ham lizat çivili ve daha sonra filtrasyon ile duruluğu . Aynı plazmid DNA, hücre lizat aşağıdaki filtreleme eklendi. Elektroforez her bir örnek (Şekil 6) hacimde yapıldı. Her numune benzer şiddette bir tek, net bant filtreleme tabanlı açıklama adımı sırasında biraz varsa DNA kaybı göstermektedir. Kültürler E. pCAT plazmid DNA içeren coli E. arınmış coli Pall Kullanıcı mcL 350 ve 1 ml DNA bağlayıcı Filtre Tabaklar, 350 mcL DNA bağlayıcı plakaların yanı sıra diğer iki marka lizatları. Üreticinin tavsiye ettiği protokolde belirtildiği gibi tüm arındırmalardan yapıldı. Yüksek DNA konsantrasyonu ve toplam verim Pall 1 ml AcroPrep Advance DNA arıtma filtresi plaka (Şekil 7 ve Tablo 2) görülür. Rakip W yüksek DNA konsantrasyonu (155 ng / ml) göstermesine rağmen, toplam DNA verimi (4.7 mikrogram / iyi) düşük kurtarma hacmi nedeniyle düşük olduğu, ~ 30 mcL. Rakip M 108 DNA düşük konsantrasyon verdi ng / toplam 5.6 mg / iyi bir verim ile mcL. Agarose jel üç farklı DNA bağlayıcı plakaları pCAT saflaştırılmış DNA analizi Şekil 8&#39;de gösterilmiştir. Her toplanan numune hacmi elektroforez önce kurtarma hacmi farklılıkları düzeltmek için 65 mcL ayarlandı. Tüm numuneler plazmid DNA supercoiled gösterir, ancak her Filtre Tabaklar DNA verimi değişir. Şekil 9 hazırlanan DNA supercoiled formu ve benzeri konsantrasyon gösterir. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Şekil 1" /> Şekil 1. Lizat netleştirilmesi için vakum Manifold Meclisi. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Şekil 2" /> Şekil 2. DNA Ciltleme için vakum Manifold Meclisi. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Şekil 3" /> Şekil 3. Vakum Elüsyon için vakum Manifold Meclisi. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Şekil 4" /> Şekil 4. Filtrasyon örnek açıklama kolaylaştırır. Vakum filtrasyonu (350 mcL AcroPrep Advance lizat açıklama filtre plakası) veya geleneksel santrifüj açıklık ham lizat 300 mcL üç nüsha örnekleri. OD 320 açıklama öncesi ve sonrası ölçülmüştür. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Şekil 5" /> Şekil 5. PDNA AcroPrep Advance lizat açıklama filtre plakası geçişi sonra tam iyileşme. 300 mcL TE tampon, 25, 50 ve 100 mikrogram / mL pCAT ile çivili. DNA konsantrasyonu ve kurtarma, K = 2 öncesi ve filtrasyon sonra OD 260 hesaplanmıştır. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Şekil 6" /> Şekil 6. Açıklama sonra pCAT DNA Kurtarma. Eşit miktarda, açıklama öncesinde veya sonrasında lizat içine çivili pUC19 sonra TE tamponunda 10 1 seyreltilmiş. % 1.2 agaroz jel üzerinde 2 mcL / şeritli yüklendi. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Şekil 7" /> Şekil 7. Pall Acroprep Advance DNA rakip plakalar daha bağlayıcı filtre plakası ile ortalama Yüksek plazmid DNA verim 1 ml (Pall ve Rakip M) veya 1.5 mL (Rakip W) bir gecede kültürü ile gösterilir DNA arıtma plakaları kullanılarak pCAT plazmid DNA verimi (OD 260) DH5α. Plaka üreticinin tavsiye protokolü kullanılarak saflaştırılması. Hata çubukları standart hata (n&gt; 6) işaret etmektedir. <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Şekil 8" /> Şekil 8. PCAT DNA agaroz jel analizi belirtilen DNA bağlayıcı plaka saflaştırılmış. Saflaştırılmış pDNA% 1.2 agaroz jel elektroforezi. Örnekleri, üç ayrı arındırmalardan Pooled eluat arıtma, seyreltilmiş 01:10 sonra 65 mcL ayarlanır. Hat başına 2 mcL yüklendi. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Şekil 9" /> Şekil 9. Kalite ve santrifüj veya vakum yöntemleri saflaştırılmış pDNA son eluat toplama agaroz jel elektroforezi için kullanılan benzer saf dna kurtarma. PDNA arıtma AcroPrep Gelişmiş Filtre Tabaklar için üreticinin tavsiye ettiği protokol izledi. Elüsyon, 2 dakika için 15 inç Hg (0,5 bar) ile 5 dakika veya vakum 1000 xg&#39;de santrifüj tarafından yapıldı.

Bu video makalenin yazarları, video ve metin özellikli cihaz ve reaktiflerin üreten Pall Yaşam Bilimleri çalışanları.

Biz çeşitli avantajları tanıyor AcroPrep Advance Filtre Tabaklar facile, aerodinamik protokol açıklar. Bu yöntem, manuel, yarı otomatik veya tam otomatik AcroPrep Advance biçimlerde işlenmiş, maksimum plazmid DNA verim sağlar. Lizat açıklık plaka üzerinde entegre bir ön filtre örnekleri, hatta partikül bu içeren yüksek düzeyde tutarlı filtrasyon sağlar. , Daha hızlı, daha düzgün bir plaka üzerinde minimal tutma hacmi ile filtrasyon hızları plaka sonuçlarının iyi geometri ve çıkış uç geometrisi, çapraz kontaminasyon endişeleri olmaksızın alıcı plakasına doğrudan numune akışı sağlar. Önemlisi, AcroPrep Gelişmiş Filtre Tabaklar DNA bağlayıcı plaka yüksek bağlama kapasitesi membran yüksek kalite plazmid DNA maksimum kurtarma için optimize edilmiştir.

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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>
		</tr>
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<tr>
<td>AcroPrep Advance Filter plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8175 (1 mL) or PN 8075 (350 &mu;l)</td>
<td>3 &mu;m glass fiber 0.2 &mu;m Supor&reg; membrane for clarification</td>
</tr>
<tr>
<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
</tr>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 13157</td>
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streamlined purification of plasmid dna from prokaryotic cultures

Biz prokaryotik kültürünün başlangıç ​​ve yüksek saflıkta DNA ile biten, 96 plazmid hazırlıkları için AcroPrep Gelişmiş Filtre Tabaklar süreci tanımlamak. Bakteriyel lizat açıklık ve DNA saflaştırma için çok iyi filtrasyon dayanarak, bu yöntem plazmid hazırlanması için akıcı bir süreç oluşturur. Silika-tabanlı medya içeren Filtre plakalar, vakum filtrasyon veya santrifüj plazmid DNA kayda değer miktarda verim kolayca işlenebilir. Nicel analiz saflaştırılmış plazmid DNA 1.97 ortalama OD 260/280 oranları ile yüksek kalitede sürekli olarak belirler. Genel olarak, sıralama ve klonlama gibi downstream uygulamalar için daha saf DNA plazmid verim sunuyoruz. Aerodinamik AcroPrep Gelişmiş Filtre Tabaklar kullanarak bu yöntem, manuel, yarı otomatik veya tam otomatik işleme sağlar.

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Bu protokol, verimli paralel açıklama ve plazmid DNA arıtma için uygun maliyetli bir alternatif<em&gt; E. coli</em&gt; Kültürler. AcroPrep Advance sürecinin yüksek bir bağlama kapasitesi DNA bağlayıcı filtre plakası arıtma ile optimize edilmiş bir lizat açıklama filtre plakası ile başlar.

Prokaryotik Kültürler itibaren Plazmid DNA Aerodinamik Arıtma

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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Video: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Plazmid DNA Qiagen Miniprep Kit kullanarak Bakteri kolonileri Arındırıcı

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Biyoloji

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Maya hücreleri Mitokondri saflaştırılması

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

İskelet Kası progenitörlerin saflaştırılması

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Önce DNA Saflaştırma olmadan Bitki ve Hayvan Örnekleri genotiplendirmesi

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

Plazmid kökenli Uygulama Kimyasal Reaksiyon Ağları için DNA Strand Deplasman kapıları

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Ökaryotik Hücrelerde gelen ekstrakromozomal dairesel DNA genom Arıtma

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...