نشكر آلان تاسكر (USDA-APHIS، ريفرديل، MD)، ستيفن كومبتون (كليمسون)، شيري Aultman (كليمسون) والويلزي كريغ رمزي (USDA-APHIS، فورت كولينز، أول أكسيد الكربون)، وبيتي Marose (الخفة) للحصول على المساعدة في الحصول على عينات . نشكر الطلاب أندرو ادريان (UA-هانتسفيل) وديريك ثاكر (UA-هانتسفيل) لمساعدتها في اختبار هذا البروتوكول، وHerdy جوزيف لعمله في تصوير الفيديو. وقد تم تمويل هذا العمل من قبل الأسماك الوطنية ومؤسسة الحياة البرية.

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+intergenic+spacer+of+ribosomal+DNA+of+Imperata+cylindrica+(L.)+Beauv.+var.+major+(Cogongrass)+populations+in+Taiwan.">Genetic variation in the intergenic spacer of ribosomal DNA of Imperata cylindrica (L.) Beauv. var. major (Cogongrass) populations in Taiwan.</a> Botanical Bulletin of Academia Sinica (Taipei). 40, 319-332 (1999).</li></ol>

ليس لدينا ما يكشف.

<html> الحضانة الولايات المتحدة والصناعات المشهد تزدهر على زراعة وبيع أنواع النباتات الغريبة ورواية. هذا، بالإضافة إلى تزايد عولمة التجارة، ويزيد من فرص أن الأنواع النباتية الغازية ستدخل، وإنشاء، وانتشرت في الولايات المتحدة القدرة على تنظيم الحكومة الفدرالية مثل هذه المصانع تعتمد على المعلومات التي غالبا ما تكون غير متوفرة، بما في ذلك القدرة على أن تصبح الغازية الصحيح والتصنيف، والطابع المميز وراثية من الأصناف المحلية والمتجنسين. لأن معرفتنا النباتات الغازية غالبا ما تكون محدودة، وقد تم النباتات المستوردة ذات الخصائص المخفية الغازية قدم عن طيب خاطر فقط لمعرفة وقت لاحق انهم غزو مواردنا الزراعية والطبيعية. هذا البروتوكول يهدف الى معالجة هذه المشاكل مرتبطة أولا أصناف cylindrica من خلال تقديم أول طريقة مبسطة الجزيئية التي يمكن أن نميز بدقة بين البرية من نوع cogongrass وشكل عادت من نظيره JBG الزينة. jove_content &quot;&gt; وبالنسبة للتنمية من هذا البروتوكول، وجمعت من النوع البري cogongrass من السكان المجنسين في وحدة البركة الغابات كريك في مقاطعة سانتا روزا بالقرب جاي، فلوريدا في شهر يونيو من عام 2008. JBG تم شراؤها من المشاتل التجارية (بلوبيرد اطفال، المؤتمر الوطني العراقي ..) في يونيو 2008 وكذلك من جمع المنزل في كولومبيا، MO JBG يعود تم الحصول عليها من باحة متحف كامبل الجيولوجي في جامعة كليمسون، واتفاقية استكهولم في يونيو 2008؛ من جامعة بارك في ريفرديل، ماجستير في يونيو 2009، و من الساحة الأمامية من المنزل في كولومبيا، مو في عام 2009 (التي تم تحديدها من قبل Cseke ليلاند). تمت المحافظة على جميع النباتات في الدفيئة التي تقع في جامعة ألاباما في هانتسفيل (هانتسفيل، AL). وشملت التسلسل الجيني للحمض النووي التي تم جمعها من هذه النباتات ومقارنات في عمق 9 مناطق الحمض النووي المستقل الذي يستخدم عادة في محطات الباركود 2. في جميع الحالات، كانت تتابع JBG مباراة بنسبة 100٪ لتلك التي تعود JBG، مما يساعد علىتحقق من أن JBG لا تعود في الواقع إلى نموذج، أخضر الغازية. فقط لبرنامجها النووي وبلاستيدات الخضراء المناطق trnL-F خلافات التي يمكن استخدامها للتمييز بين وراثيا cogongrass وJBG. في منطقته لديها ما مجموعه 3 تعدد الأشكال (الأشكال المتعددة للتركيبات النووية المنفردة) بين cogongrass وJBG، في حين ان المنطقة trnL-F قد 2 و 2 النيوكلوتايد InDels (الإدراج والحذف). هذه الاختلافات الوراثية سمح متنوعة محددة بادئات PCR إلى تطوير التي يمكن أن تميز بين من النوع البري cogongrass ويعود JBG. قد تأتي النتائج الأكثر موثوقية من الاشعال المستمدة من المنطقة trnL-F صانعة. وهكذا، ويستند هذا البروتوكول على الفوارق بين المناطق تسلسل trnL-F من الجينوم بلاستيدات الخضراء من cogongrass، JBG وعادت JBG (الشكل 4). للمساعدة في توليد الاشعال التي هي أكثر تحديدا للأصناف المذكورة والمساعدة على تجنب ايجابيات كاذبة من أنواع وثيقة الصلة، آلل معروف trnL-F تسلسل أولا وتمت مقارنة مع أصناف cylindrica trnL-F متواليات من أنواع الأعشاب ذات الصلة (43 سلاسل مستقلة من 29 نوعا، على سبيل المثال، Cymbopogon citratus، Sorghastrum incompletum، دمع أيوب، jobi، Miscanthus سينينسيس، قصب السكر officinarum، halepense الذرة البيضاء). وعلى الرغم من أننا فحص متواليات التمهيدي متنوعة محددة عبر 29 نوعا من العشب، وخصوصية الاشعال تشكيلة معينة لم يتم دراستها على نحو شامل لقدرته على تضخيم الحمض النووي من معظم الأنواع الأخرى العشب. وبناء على ذلك، ينبغي أنسجة المستخدمة لاستخراج الحمض النووي التي تم تحديدها بعناية كما I. cylindrica قبل البدء في هذا البروتوكول. إذا لا يمكن تحديد العشب إما cogongrass أو JBG، ثم نقترح التسلسل للمنتج PCR للتأكد من أن تسلسل وتطابق تام إلى cogongrass أو JBG. حاليا، فإن الطريقة الأكثر دقة للتحقق من هوية من عينة عشب معين هو لأداء PCR في تي في على حد سواءrnL-F والمناطق، تليها سلسلة من المنتجات والتحقق PCR ومقارنة تسلسل إلى تسلسل معروف من الأنواع التي تم تحديدها بدقة. ويمكن تضخيم الحمض النووي باستخدام بادئات المراقبة المفصلة في هذا البروتوكول (لمنطقة trnL-F) أو الاشعال الأخرى التي تتوافر في غيرها من المنشورات 13-15. التسلسل هو عمل أكثر من ذلك بكثير وتكلفة مكثفة من استخدام إجراءات مبسطة لدينا. نوعية الاشعال PCR تستخدم لأمر بالغ الأهمية لنجاح هذا الإجراء. لقد جعلنا من الاشعال لهذا الإجراء المتاحة من شركة المائل، بيو ( <a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> ، هانتسفيل، AL). ميزة ليأمر الاشعال من بيو المائل هي أنها تخزين أعداد كبيرة من aliquots من كل التمهيدي من سلسلة إنتاج نموذج مطابق كما الاشعال نحن تستخدم لتعظيم الاستفادة في مختبرنا. وبناء على ذلك، الاشعال ليس فقط تسلسل نفسه، ولكن تيمهلا تأتي من الكثير إنتاج نفسها بالضبط التي تم استخدامها في هذا البروتوكول. باستخدام بادئات من نفسه الكثير، يمكن أن تساعد على تجنب المتغيرات الدخيلة في الإجراء الذي قد ينجم عن الاختلاف في نوعية بادئات PCR. وعلى نحو مماثل، في حين الأخرى بلمرة طق ينبغي أن تعمل بشكل جيد لPCR، ونوعية بوليميريز طق المستخدمة لها تأثير على جودة نتائج PCR. للسماح لأفضل الاتساق في الكواشف PCR، ونحن الأمثل للبروتوكول باستخدام الكواشف من Clontech. وميزة 2 بوليميريز (Clontech، ماونتن فيو، كاليفورنيا، القط # 639201 أو 639202) هو خليط من قوية، بوليميريز طق الساخنة البداية وانزيم تصحيح التجارب المطبعية التي تساعد على تقديم نوعية عالية والتكبير أكثر دقة. لأن هذا البروتوكول يعتمد على الحمض النووي بلاستيدات الخضراء، التي ورثت أمومي في الأعشاب، قد لا أحداث التهجين بين cogongrass والأنماط الجينية JBG يتم القبض مع إجراء لدينا تحديد الجزيئية. في الحالات التي يكون فيها الجسمى هو suspeالمديرية، ونحن نوصي باستخدام مناطق النووية التي ورثتها من الآباء والأمهات على حد سواء. الأكثر استخداما غير صانعة منطقة المتغير للنظر في التنميط الجيني للنبات هو النووي الريباسي ITS منطقة 13-15،17. حاليا، نحن نحرز تقدما نحو الإرسال المتعدد التضخيم من المنطقة trnL-F بلاستيدات الخضراء مع أن المنطقة النووي في أنبوب PCR نفسه. والإرسال المتعدد صانعة مع مناطق الحمض النووي الالتفاف المحتمل أن القيود المفروضة على استخدام إما وحدها، ولكن مثل هذه الأساليب تتطلب التحسين وتقييم إضافية لتحديد الجدوى الاقتصادية على أساس كل حالة على حدة. كما أن استخدام PCR في الوقت الحقيقي الكمية (qPCR)، والتكنولوجيات الجديدة، مثل منارات الجزيئية (تحقيقات اولية فلوري)، كما يجري حاليا تقييم آمنة من الفشل ودقة أساليب التنميط الجيني للنبات. بروتوكول المقدمة هنا يقدم نهجا سريعة وموثوق بها للتمييز بين JBG تعود من أن من البرية من نوع cogongrass. نحن نشجع استخدامالتمرير من هذا البروتوكول في الاتصال بنا للإبلاغ النتائج المستمدة من استخدام هذا البروتوكول. لا بل ان هذه المعلومات المشتركة تساعد على توفير معلومات عن توزيع JBG يعود. وسيساعد هذا أيضا المنظمين في وزارة الزراعة الأميركية من اتخاذ قرارات مستنيرة بشأن الإجراءات التي قد تكون في حاجة للالتفاف على انتشار والتهجين المحتملة للأصناف cogongrass الغازية للغاية.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> DNeasy النبات البسيطة كيت </td><td> QIAGEN، فالنسيا، كاليفورنيا </td><td> 69104 أو 69106 </td><td> وينبغي أن أي مصنع الجينومية السمعة عدة الحمض النووي DNA يمكن أن ينتج جيدة الجودة تعمل بشكل جيد لهذه الإجراءات. </td></tr><tr><td> trnF (GAA)-F </td><td> منحرف بيو، وشركة </td><td> 3-0578 </td><td> إيجابية إلى الأمام مراقبة التمهيدي </td></tr><tr><td> trnL (إكسون 5 &#39;)-C </td><td> منحرف بيو، وشركة </td><td> 3-0579 </td><td> عكس إيجابي التمهيدي مراقبة </td></tr><tr><td> trnLF-C-F1 </td><td> منحرف بيو، وشركة </td><td> 3-0864 </td><td> التمهيدي إلى الأمام cogongrass من النوع البري </td></tr><tr><td> trnLF-C-R1 </td><td> منحرف بيو، وشركة </td><td> 3-0865 </td><td> عكسمن النوع البري cogongrass التمهيدي </td></tr><tr><td> trnLF-R-F2 </td><td> منحرف بيو، وشركة </td><td> 3-0866 </td><td> JBG إلى الأمام وJBG تعود التمهيدي </td></tr><tr><td> trnLF-R-R2 </td><td> منحرف بيو، وشركة </td><td> 3-0867 </td><td> عكس JBG وJBG تعود التمهيدي </td></tr><tr><td> ميزة عالى النقاء PCR dNTP ميكس </td><td> Clontech، ماونتن فيو، كاليفورنيا </td><td> 639125 </td><td></td></tr><tr><td> ميزة 2 البوليميراز </td><td> Clontech، ماونتن فيو، كاليفورنيا </td><td> 639201 أو 639202 </td><td> وخير دليل على القراءة، بوليميريز طق الساخنة البداية </td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

ونحن نقدم فعالة من حيث التكلفة وسريعة بروتوكول التنميط الجيني التي توظف متنوعة محددة الاشعال PCR أن الاختلافات الهدف تسلسل الحمض النووي داخل المنطقة هل بلاستيدات trnL-F للتمييز بين أنواع مختلفة من<em&gt; Imperata cylindrica</em&gt; (cogongrass) التي لا يمكن تمييزها عن طريق التشكل وحدها. هذه الأصناف وتشمل الأعشاب الضارة الواردة اتحاديا، cogongrass وثيقة الصلة، واسعة الانتشار متنوعة الزينة، وأنا<em&gt;. cylindrica</em&gt; فار.<em&gt; koenigii</em&gt; (يابانية عشب الدم).

والطريقة انماط PCR القائم على التمييز بين الأنواع البرية من نوع والزينة لل Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

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Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.4K Views.  The University of Alabama, Huntsville. ونحن نقدم فعالة من حيث التكلفة وسريعة بروتوكول التنميط الجيني التي توظف متنوعة محددة الاشعال PCR أن الاختلافات الهدف تسلسل الحمض النووي داخل المنطقة هل بلاستيدات trnL-F للتمييز بين أنواع مختلفة من Imperata cylindrica (cogongrass) التي لا يمكن تمييزها عن طريق التشكل وحدها...

Video: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : طريقة بسيطة لالتنميط الجيني دقيقة

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

تحديد آثار الطفرات BRCA1 على التوحد باستخدام خلايا متماثلة التي تعبر عن الذاتية وحشية من نوع BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

الفحص البروتين تراكب الأغشية : بروتوكول لاختبار التفاعل بين البروتينات القابلة للذوبان وغير القابلة للذوبان في المختبر</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

PCR الأمثل القائم على الكشف عن المفطورة

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

الايس كاب : منهج في تزايد Arabidopsis الطماطم والنباتات في لوحات 96 - جيدا لالرفيع المناولة التنميط الجيني

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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.

التنميط الجيني للعينات النباتية والحيوانية دون تنقية DNA قبل

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

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium الفحص على نطاق واسع لتطبيقات SNP التنميط الجيني

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

سريعة وفعالة الزرد التنميط الجيني باستخدام PCR عالية الدقة مع تحليل نذوب

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

تحليل qPCRTag - إنتاجية عالية، في الوقت الحقيقي PCR الفحص لSc2.0 التنميط الجيني

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

تحديد الأحماض السياليك في الكبد وعينات الحليب من البرية من نوع و<em&gt; CMAH</em&gt; خروج المغلوب الفئران.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...