1. Лизат оформление <ol><li> Расти Е. кишечной культур трансформированных желаемого плазмидной ДНК в глубокий колодец пластин (1 мл культуры / лунку) в бульоне Лурия с соответствующими антибиотиками в течение ночи при 37 ° C при встряхивании. </li><li> Гранул кишечная палочка в культуре табличку на 5000 мкг в течение 10 минут, затем перелить супернатант. </li><li> Ресуспендируют каждая гранула в 100 мкл буфера ресуспендирования (50 мМ Трис-HCl, рН 8,0, 10 мМ ЭДТА, 100 мкг / мл РНКазы). </li><li> Добавить 100 мкл / лунку Лизис буфера (200 мМ NaOH, 1% SDS) и встряхните использовании планшетном шейкере в течение 2 минут. </li><li> Добавить 100 мкл / лунку Нейтрализация буфера (3,0 М ацетата калия, рН 5,5) и встряхивают в течение 2 минут. </li><li> Передача Клеточный лизат к лизат разъяснения пластины. </li><li> Место связывания ДНК пластины сверху 350 мкл пластины коллекцию и поместить в нижней части вакуумного многообразия. Сборка вакуумного многообразия (см. Рисунок 1). </li><li> Место лизат разъяснения пластину на верхней части вакуумного аппарата. </li><li> Применение вакуума при 10 дюймов Hg (0,34 бар) и сбор фильтрата в связывания ДНК пластины. Не используйте вакуум больше 12 дюймов рт. (0,41 бар). </li><li> Разберите вакуумного многообразия. Место 2 мл сбора отходов пластина в нижней части аппарата. </li></ol> 2. Связывание ДНК <ol><li> Перемещение связывания ДНК пластины в начало многообразии (см. Рисунок 2). </li><li> Добавить 300 мкл / лунку буфера для связывания (6 М гуанидин-HCl) и пипетка вверх и вниз, чтобы смешаться. </li><li> Применение вакуума [~ 5 дюймов Hg (0,17 бар) для медленных вакуум] и отбросить фильтрата. ДНК теперь связан с мембраной. </li><li> Промыть 400 мкл / лунку промывочного буфера (80% этанола). </li><li> Применение вакуума, затем удалите фильтрата. Удалите ненужные, если используются 2 мл коллекции плита или фильтрации непосредственно в отходы. </li><li> Повторите мыть один раз. </li><li> Пятно нижней части фильтра пластина на впитывающее полотенце для просушки. </li><li> Применение вакуумных еще раз в течение 5-10 минут, чтобы обеспечить удаление следов спирта. </li><li> Пятно снизу обеспечить удаление этанола капель. </li></ol> 3. ДНК Элюирование <ol><li> Добавить 70 мкл / лунку элюции буфера (10 мМ Трис-HCl, рН 8,0, 1 мМ ЭДТА). Если выше концентрации ДНК желательно, объем может быть уменьшен до 50 мкл / лунку, но в среднем общий выход будет ниже. </li><li> Инкубируйте планшет при комнатной температуре в течение одной минуты. </li><li> Очищенная ДНК может быть элюировали либо вакуумом или центрифугирования. </li><li> Вакуумный метод: Поместите чистую тарелку коллекции (ДНКазы, РНКазы бесплатно) в вакуум многообразия. Место фильтр пластину на верхней части вакуумного многообразия, применять вакууме при 15 дюймов Hg (0,5 бар) в течение 1 минуты, пока все элюции буфера прошла через связывания ДНК пластины. Сбор очищенную ДНК (см. рисунок 3). </li><li> Центрифугирование Метод: Положите Фильтр для очистки пластины сверху чистую тарелку сбора и центрифуге при 1000 мкг в течение 5 минут. </li></ol> 4. Количественный и качественный анализ <ol><li> Мера OD 260/280, рассчитать концентрацию очищенной ДНК. </li><li> Выполните агарозном геле анализ очищенного плазмидной. </li></ol> 5. Представитель Результаты Кишечная палочка лизат уточнены с использованием традиционных центрифугирования и Pall AcroPrep Advance лизат разъяснения фильтра пластины показали заметное сокращение мусора из исходного материала. (Рис. 4). ДНК плазмиды в трех концентрациях шипами в ТЕ буфере и проходил через лизат AcroPrep Advance разъяснения фильтр пластины. Сравнение концентрации ДНК плазмиды, пре-и пост-фильтрации, показывает ~ 100% восстановления (рис. 5) на всех трех концентрациях. Чтобы продемонстрировать восстановление ДНК из уточнил Е. кишечной лизат, плазмиды ДНК шипами в сырой лизат, а затем уточнить путем фильтрации. Же ДНК плазмиды был добавлен в ячейке фильтрации следующие лизат. Электрофорез проводили на аликвоты из каждого образца (рис. 6). Единый, ясный полосы одинаковой интенсивности из каждого образца демонстрирует практически никакой ДНК потерь при фильтрации основе разъяснения шаг. Культуры E. кишечной содержащие рСаЬ плазмидной ДНК очищали от E. кишечной лизатов использованием 350 Полла мкл и 1 мл связывания ДНК фильтра плиты, а также два других марок 350 мкл связывания ДНК пластин. Все очищения были выполнены, как указано в протоколе рекомендуется производителем. Самая высокая концентрация ДНК и полный выход показаны с 1 Полла мл AcroPrep Advance ДНК Фильтр для очистки пластин (рис. 7 и таблицу 2). Хотя конкурентов W также показывает высокую концентрацию ДНК (155 нг / мкл), суммарный выход ДНК является самым низким (4,7 мкг / лунку) в связи с низким объемом восстановления, ~ 30 мкл. Конкурент М представил самую низкую концентрацию ДНК в 108 нг / мкл с общим выходом 5,6 мкг / лунку. АгарOSE гель анализ очищенного рСаЬ ДНК из трех различных связывания ДНК пластин показано на рисунке 8. Каждый собранный объем пробы доводили до 65 мкл для корректировки различий в восстановлении объема до электрофореза. Все образцы показывают суперспиральной плазмидной ДНК, но ДНК доходность от каждого из фильтровальных плит варьируется. На рисунке 9 показан подготовленный ДНК все в суперспиральной форме и имеет такие же концентрации. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Ассамблея Вакуумный коллектор для лизат разъяснение. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Рисунок 2" /> Рисунок 2. Ассамблея Вакуумный коллектор для связывания ДНК. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Рисунок 3" /> Рисунок 3. Ассамблея Вакуумный коллектор для вакуумной элюции. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Рисунок 4" /> Рисунок 4. Фильтрация упрощает образец разъяснения. Triplicate образцы 300 мкл сырой лизат осветляли вакуумной фильтрации (350 мкл AcroPrep Advance лизат разъяснения фильтр пластины) или традиционным центрифугирования. OD 320 измеряется до и после разъяснения. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Рисунок 5" /> Рисунок 5. Полное восстановление плазмидной после прохождения AcroPrep Advance лизат пластины фильтра разъяснения. 300 мкл буфера TE подсыпали рСаЬ в 25, 50 и 100 мкг / мл. Концентрация ДНК и восстановления рассчитывается OD 260 до и после фильтрации, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Рисунок 6" /> Рисунок 6. Восстановление рСаЬ ДНК после разъяснения. Равные количества pUC19 шипами в лизат до или после разъяснений, затем разбавляют 1 до 10 в буфере TE. Загружено 2 мкл / полосы на 1,2% агарозном геле. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Рисунок 7" /> Рисунок 7. Высшее плазмиды выход ДНК на лунку с покров Acroprep Advance связывания ДНК фильтр пластины, чем конкурент пластин. РСаЬ плазмиды выход ДНК (OD 260) с использованием указанных пластин очистки ДНК с 1 мл (Pall и Конкурент М) или 1,5 мл (Конкурент Вт) в течение ночи культуры DH5α. Очистка с использованием рекомендованных протокол пластины производителя. Ошибка полоски показывают стандартную ошибку (я&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Рисунок 8" /> Рисунок 8. Агарозном геле анализ ДНК рСаЬ очищали на указанные связывания ДНК пластины. 1,2% агарозном гель-электрофорез очищенного плазмидной. Собранные пробы из трех отдельных очищения, элюата доводили до 65 мкл после очистки, разбавленный 1:10. Загружено 2 мкл на одну полосу движения. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Рисунок 9" /> Рисунок 9. Качество и восстановление чистой ДНК подобное, когда центрифуга или вакуумные методы используются для окончательного элюата коллекции агарозном гель-электрофорез очищенного плазмидной. Рекомендуется протокол производителя AcroPrep Advance Пластины фильтра последовали для очистки плазмидной ДНК. Элюирование исполнении центрифуге при 1000 мкг в течение 5 минут или вакуум с 15 дюймов Hg (0,5 бар) в течение 2 минут.

Авторы этого видео-статьи, сотрудники Pall Life Sciences, кто производит инструмент и реагенты в ролике и текст.

Мы описываем поверхностное, обтекаемый протокол пластин AcroPrep Advance фильтра, который предоставляет ряд преимуществ. Этот метод обеспечивает максимальную доходность плазмиды ДНК, когда обрабатывается в ручном, полуавтоматическом или полностью автоматизированных форматов AcroPrep Advance. Интегрированный фильтр предварительной очистки на лизат пластины клиренс обеспечивает постоянную фильтрацию образцы, даже те, с высоким содержанием твердых частиц. И геометрия пластины приводит к более быстрой, более равномерное скорости фильтрации через пластины с минимальным ограбления объем и геометрию выходе наконечник обеспечивает прямой поток образцов в приемнике пластиной без проблем перекрестного загрязнения. Важно отметить, что высокие обязательные мембраны потенциала связывания ДНК пластины AcroPrep Advance Пластины фильтра оптимизирована для максимального извлечения высококачественной плазмидной ДНК.

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		<div>
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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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>
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<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

Мы опишем полный процесс AcroPrep Advance фильтра Плиты для 96 плазмиды препаратов, начиная от прокариотических культуры и заканчивая высокой чистоты ДНК. На основе нескольких хорошо фильтрации для бактериальной очистки лизата и очистки ДНК, этот метод создает упрощенный процесс с плазмидной ДНК. Фильтры чашек, содержащих кремний-СМИ может быть легко обработан вакуумной фильтрации или центрифуги для получения заметных количеств плазмидной ДНК. Количественный анализ определить очищенной ДНК плазмиды неизменно высокого качества со средним диаметром 260/280 отношение 1,97. В целом, плазмиды дает предлагают более чистой ДНК для последующих приложений, таких как секвенирование и клонирования. Этот обтекаемый метод использования AcroPrep Advance фильтра пластин позволяет для ручной, полуавтоматический или полностью автоматизированной обработки.

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

Этот протокол представляет собой экономичную альтернативу для эффективных параллельных разъяснения и плазмиды очистки ДНК из<em&gt; Е. палочки</em&gt; Культур. Процесс AcroPrep Advance начинается с оптимизированным лизат разъяснения фильтр пластины с последующей очисткой на высокой связывающей способности связывания ДНК фильтр пластины.

Упрощенная очистка плазмидной ДНК из Прокариотические культур

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.

Очищающий плазмидной ДНК из бактериальных колоний Использование 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 ...

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.

Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

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.

Окрашивание белков в гелях Кумасси G-250 без органических растворителей и уксусной кислоты

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.

Очистка Митохондрии из дрожжей Клетки

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.

Очистка прародителей из скелетных мышц

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

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Выделение и очистка от кинезин Drosophila Эмбрионы

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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.

Генотипирование растений и животных образцов без предварительного очистки ДНК

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.

Субстрат поколения для эндонуклеаз CRISPR / Cas системы

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.

Плазмиды, полученные ДНК замещения цепи Гейтс для реализации химической реакции сети

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 ...

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.

Генома Очистка экстрахромосомального кольцевой ДНК из эукариотических клеток

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