وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح GM45916 وGM66834 إلى JCH وزمالة من المؤسسة الدولية ريت متلازمة لحزب العدالة والتنمية هذا العمل كان مدعوما أيضا من قبل المعاهد الوطنية للصحة منح GM088409 ومعهد هوارد هيوز الطبي المساهمات في كوالا لمبور

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نموذج صفائف النووي nucleosomal هي أداة مفيدة جدا لدراسة في المختبر من هيكل لونين وظيفة. على سبيل المثال، فقد استخدمت على نطاق واسع لدراسة آلية الألياف لونين التكثيف في حل 30-34، وجعلت من الممكن للحصول على هيكل الأشعة السينية من tetranucleosome 35. في الآونة الأخيرة ا...

الجينوم حقيقية النواة لا وجود الحمض النووي عارية كما، بل يتم ضغط ونظمته البروتينات ملزمة. وتعرف هذه المجمعات من الحمض النووي والبروتينات من لونين. الوحدة الأساسية للمكرر لونين هو جسيم نووي. يتكون جسيم نووي من octamer هيستون و 146 أزواج قاعدة الحمض النووي ملفوفة حول octamer هيستون نحو 1.6 مرات 1. يتكون octamer هيستون من نسختين كل من H2A histones الأساسية، H2B، H3، H4 و. وتسمى octamers هيستون الأساسية التي يتم متباعدة بشكل متكرر على طول جزيء الحمض النووي صفائف النووي nucleosomal. وقد أشار هيكل ممتدة من صفائف النووي nucleosomal على أنها الألياف 10 نانومتر أو &quot;الخرز على سلسلة&quot; هيكل وموجود في المختبر تحت ظروف الملح منخفضة 2. الألياف 10 نانومتر قادر على التكثيف في هياكل النظام من خلال الضغط العالي داخل مجموعة و / أو بين مجموعة oligomerization 2. هذه الهياكل أعلى لكي يمكن أن يتسبب في وجود الأملاح،أو يمكن أن تتأثر من خلال الربط من البروتينات المعمارية لونين لمجموعة النووي nucleosomal 3،4. ترتبط مستويات ونين الضغط عكسيا مع معدل النسخ في الجسم الحي 5،6. يسلط الضوء على البحوث التي أجريت مؤخرا على أهمية التنظيم الهيكلي من الجينوم في عمليات مثل التمايز، تطور مرض السرطان، وغيرهم 7،8. أصبح استخدام صفائف النووي nucleosomal لدراسة بنية الكروماتين وظيفة على نطاق واسع. نحن هنا تصف طريقة لتجميع صفائف النووي nucleosomal من histones الأساسية المؤتلف الحمض النووي وتحديد المواقع جسيم نووي. باستخدام الحمض النووي المؤتلف مع يكرر جنبا إلى جنب من تسلسل المواقع النيوكليوسومات يسمح لإعادة تشكيل المصفوفات التي تحتوي على Nucleosomes للمتباعدة بصورة منتظمة. اثنين من متواليات تحديد المواقع أكثر شعبية هي تسلسل الجين 5S الريباسي و &quot;601&quot; 9،10 التسلسل. تم اشتقاق تسلسل 601 من التجارب سيليكس وموره المواقف بقوة Nucleosomes للمن تسلسل 5S 11. وبالتالي فإن طول الحمض النووي رابط من صفائف 601 هو أكثر تجانسا. يتم الحصول على الحمض النووي المتكررة Tandemly المواقع النيوكليوسومات بواسطة هلام 4،12 الترشيح. يتم تنقيته الهستونات المؤتلف من E. القولونية في ظل ظروف تغيير طبيعة 13. استخدام الهستونات المؤتلف يسمح احد للسيطرة بعناية تكوين هيستون من صفائف النووي nucleosomal. على سبيل المثال، الأساسية الهستونات تحمل طفرات محددة 14 أو التعديلات بعد متعدية 15،16 يمكن أن تكون بديلا عن النوع البري histones الأساسية. تجارب الترسيب سرعة رصد معدل الترسيب من الجزيئات في محلول تحت قوة الطرد المركزي التطبيقية 17. هذا يعطي معلومات حول حجم وشكل الجزيئات في عينة. تجارب الترسيب السرعة وبالتالي أداة مناسبة لدراسة التغيرات في حل الدولة ألياف الكروماتينهيكل بسبب التكثيف لونين 18. الأهم من ذلك، فمن الضروري أولا لاستخدام الترسيب التجارب سرعة كخطوة لمراقبة الجودة في مجموعة إعادة النووي nucleosomal. إذا لم يتم الجمع بين الحمض النووي وNucleosomes للفي نسبة المولي الصحيح، قد يكون صفائف أقل أو أكثر مشبعة histones الأساسية. وبالتالي، يتم استخدام المعلومات المكتسبة من التجارب سرعة الترسيب لضمان أن الحمض النووي غير المشبعة بشكل صحيح مع Nucleosomes لل. من المهم استخدام طرق بديلة لتقدير التشبع من الحمض النووي مع Nucleosomes لل، وخاصة إذا كان يعمل مع قالب الحمض النووي uncharacterized سابقا. لذلك، نحن أيضا تصف طريقة لتحليل المصفوفات النووي nucleosomal باستخدام القوة الذرية المجهري (AFM). فؤاد هو أسلوب القوية التي تسمح برؤية آثار عدد من المعلمات، مثل مستوى التشبع، تأثير وجود متغيرات هيستون أو آثار MgCl 2 19،20. وكان فؤاد أيضا تطبيق صحيفةإد لدراسة ديناميات النيوكليوسومات باستخدام الفاصل الزمني التصوير 21. تجميعها في المختبر النووي nucleosomal صفائف 12 مير قابلة خاص للدراسات فؤاد لأنها تنتمي في مجموعة الحجم المناسب للتصوير فؤاد 22. في هذه الدراسة وقد استخدمنا فؤاد صفائف النووي nucleosomal باعتباره مراقبة الجودة وكذلك وسيلة لتأكيد البيانات من الجامعة الأمريكية بالقاهرة (&quot;المشاهدة خير برهان&quot;). بالإضافة إلى التصور بسيطة، فؤاد يسمح قياس ملامح ارتفاع عينات بوصفها متري إضافية.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(3-Aminopropyl)triethoxysilane</td>
 <td>Sigma-Aldrich </td>
 <td>A3648-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>6-8 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>21-152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>HiLoad Superdex 200 16/60 Column</td>
 <td>GE</td>
 <td>17-1069-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Vivaspin 50 kDa MWCO Centrifugal Concentrator</td>
 <td>Sartorius</td>
 <td>VS2031</td>
 <td></td>
 </tr>
 <tr>
 <td>12-14 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>08-667A</td>
 <td></td>
 </tr>
 <tr>
 <td>Illustra Sephacryl S-1000 Superfine</td>
 <td>GE</td>
 <td>17-0476-01 </td>
 <td></td>
 </tr>
 <tr>
 <td>XL-A/I Analytical Ultracentrifuge</td>
 <td>Beckman-Coulter</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna

وتسمى octamers هيستون الأساسية التي يتم متباعدة بشكل متكرر على طول جزيء الحمض النووي صفائف النووي nucleosomal. ويتم الحصول على صفائف النووي nucleosomal في واحدة من طريقتين: تنقية من مصادر في الجسم الحي، أو إعادة في المختبر من histones الأساسية المؤتلف والحمض النووي المتكررة tandemly النيوكليوسومات تحديد المواقع. الأسلوب الأخير له فائدة السماح لتجميع موحدة أكثر إنشائي ومجموعة النووي nucleosomal ضعه على وجه التحديد. تجارب الترسيب السرعة في المعلومات التحليلية العائد نابذة فائقة السرعة حول حجم وشكل الجزيئات من خلال تحليل المعدل الذي تهاجر من خلال حل تحت قوة الطرد المركزي. هذه التقنية، جنبا إلى جنب مع القوة الذرية المجهر، ويمكن استخدامها لمراقبة الجودة، وضمان أن غالبية قوالب الحمض النووي ومشبعة Nucleosomes للإعادة بعد. نحن هنا تصف البروتوكولات اللازمة لإعادة كميات مليغرام من طول ود إنشائيصفائف النووي nucleosomal efined مناسبة للدراسات الفيزيائية الحيوية والكيمياء الحيوية وهيكل لونين وظيفة.

لتوضيح البروتوكول وأعيد صفائف النووي nucleosomal من المؤتلف histones الأساسية القيطم والحمض النووي يتكون من 12 جنبا إلى جنب 207 يكرر غليان من تسلسل 601 لتحديد المواقع (601 207 × 12). اجتمعنا أول octamers الأم من histones الأساسية مجفف بالتجميد ثم تنقيته من octamers بواسطة FPLC باستخدام عم...

Watch this Scientific Journal Video about Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA at JoVE.com

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

ويرد طريقة لإعادة تشكيل نموذج صفائف النووي nucleosomal من histones الأساسية المؤتلف والحمض النووي المتكررة tandemly النيوكليوسومات تحديد المواقع. نحن تصف أيضا كيفية استخدام التجارب الترسيب السرعة في نابذة فائقة السرعة التحليلية، والقوة الذرية المجهري (AFM) لمراقبة مدى النووي nucleosomal مجموعة التشبع بعد إعادة.

جمعية النووي nucleosomal صالحة من المؤتلف الأساسية الهستونات وجسيم نووي DNA لتحديد المواقع

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

assembly-nucleosomal-arrays-from-recombinant-core-histones-nucleosome

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Anatomy and Physiology

Biology

Unit 1

Cells and their Components

SCIENTISTS IN ACTION

22.4K Views.  Colorado State University.  ويرد طريقة لإعادة تشكيل نموذج صفائف النووي nucleosomal من histones الأساسية المؤتلف والحمض النووي المتكررة tandemly النيوكليوسومات تحديد المواقع. نحن تصف أيضا كيفية استخدام التجارب الترسيب السرعة في نابذة فائقة السرعة التحليلية، والقوة الذرية المجه?...

Video: Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Isolation of Genomic DNA from Mouse Tails

عزل الحمض النووي الجينوم من الذيول ماوس

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

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

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

الجمع بين QD - الحنق و microfluidics الحمض النووي لرصد Nanocomplex الذاتي الجمعية في الوقت الحقيقي

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

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

التجمع ، والتحميل ، والمحاذاة لخلية نموذج تحليلي نابذة فائقة السرعة

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

استخراج عالية الوزن الجزيئي الحمض النووي الجينوم من التربة والرواسب

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

انتقائية لقطة من hydroxymethylcytosine-5 من الحمض النووي الجيني

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

تصميم واستخدام المصفوفات ناظم كيميائي تعدد في الإرسال

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

كفاءة الإنتاج وتنقية المؤتلف الفئران Kindlin-3 من خلايا الحشرات للدراسات البيوفيزيائية

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

التصوير في الوقت الحقيقي من واحدة المهندسة رنا النصوص في الخلايا الحية عن طريق Ratiometric ثنائي الجزيء منارات

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

تنقية الكروماتوغرافي من غير البروتينية المؤتلف الإيلاستين مثل والإنشطار مع الببتيدات والبروتينات من<em&gt; كولاي</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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.

التضخيم، الجيل التالي من التسلسل، والحمض النووي الجينوم رسم خرائط مواقع التكامل فيروسات الرجعية

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