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Guest IP-штамп: fr45uHVgO7WZ2 гость |
а потом подымаеш полимеразу |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
нужно РЯДЫШКОМ сравнивать |
ssve Участник |
(Tom1 @ 04.12.2006 18:40) "а я все и процессе ПЦР вижу, еще до окончания реакции, и за минимальную стоимость" Имхо видит то что надо клиенту...... Вы не поняли сказано же все на глаз....... Есть ощущение, что вы ПЦР в реальном времени никогда не видели и не знаете, что это такое! Представьте себе, что я вижу всю кинетику прямо в процессе реакции, именно на глаз! и не надо ломать голову, та плосочка или не та! |
ssve Участник |
(Guest @ 04.12.2006 18:38) 2 ssve - у вас концы с концами того - с одной стороны ОПТИМИЗИРУЕТЕ до опупеню то есть ФОРЕЗЫ СТАВИТЕ с другой стороны КОМНАТЫ НЕТУТИ Комната есть, только оптимизирую я один раз в самом начале, а потом счастливо пользуюсь этим, не заходя в форезную ! |
ssve Участник |
(Guest @ 04.12.2006 19:17) Это у Вас с мультиплексами тое, если Вы ими не пользуетесь одной полимеразой много не добьешься |
ssve Участник |
(Guest @ 04.12.2006 19:01) 2 ssve - это для поднятья ентуазизму с ТакМанами 1: Mol Cell Probes. 2000 Apr;14(2):57-60.Click here to read Links Influence of DNA polymerases on quantitative PCR results using TaqMan probe format in the LightCycler instrument. * Kreuzer KA, * Bohn A, * Lass U, * Peters UR, * Schmidt CA. К счастью, я это знаю уже много лет, так что Америку Вы не открыли Я и не утверждала, что можно взять самые дешевые реактивы, обычную полимеразу и сразу же получится мультиплекс Как раз качество реактивов надо контролировать очень тщательно и будет вам счастье |
ssve Участник |
(Guest @ 04.12.2006 19:08) 2 ssve - кошку находят стандартным ПЦР с этидьБРОМИДОЙ и тока ПОТОМ пробуют провести РЕАЛ-Тайм - если НУЖНО Это мифы, придуманные любителями этидьБРОМИДА и джина. Если в лаборатории стоит риал-тайм для количественного анализа, то намного удобней и дешевле проводить качественный анализ на нем же |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
форезную статейку я вам кинул от "мировых лидеров" в ПЦР -скрининге а вы и не заметили на одном циклере ребята делают 1054 ваших триплексов за 1 час полтора и 10 минут форез да и работают они не ветлечебнице или станции переливания крови поетому ну не уболтаете вы меня на АНК-32 или ДТ-322 "ТакМан-скреенинг" |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
1: Nucleic Acids Res. 2005 Aug 8;33(14):e126.Click here to read Click here to read Links Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products. * Shigemori Y, * Mikawa T, * Shibata T, * Oishi M. Kazusa DNA Research Institute 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products. |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
ни обычной Тетрады на 364- 1054 реакций нету А есть один АНК-16 ( 32-может ) на которого Вас "повысили" |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
плавали-знаем |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
приходится у других спрашивать 1: Anal Biochem. 2005 Sep 1;344(1):33-42.Click here to read Links Four-color multiplex reverse transcription polymerase chain reaction--overcoming its limitations. * Persson K, * Hamby K, * Ugozzoli LA. Gene Expression Division, Bio-Rad Laboratories, Hercules, CA 94547, USA. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted in real time is a powerful tool for measuring messenger RNA (mRNA) levels in biological samples. Multiplex PCR is defined as the simultaneous amplification of two or more DNA (cDNA) targets in a single reaction vessel and may be carried out only using uniquely labeled probes for each target. Up to four genes can be detected in a multiplex 5' nuclease assay when using the appropriate instrument and the right combination of fluorophores. One of the more important advantages of multiplexing is a reduced sample requirement, which is especially important when sample material is scarce. Additional benefits are saving time on reaction setup and lower cost compared to singleplex reactions. Although multiplexing has several advantages over singleplex qRT-PCR, limited work has been done to show its feasibility. Few publications on four-color multiplex qRT-PCR have been reported, and to our knowledge no work has been done to explore the assay's limitations. In this paper, we report the first in-depth analysis of a four-gene multiplex qRT-PCR. To achieve a better understanding of the potential limitations of the qRT-PCR assay, we used in vitro transcribed RNA derived from four human genes. To emulate gene expression experiments, we developed a model system in which the in vitro transcripts were spiked with plant total RNA. This model allowed us to develop an artificial system closely resembling differential gene expression levels varying up to a million fold. We identified a single "universal" reaction condition that enabled optimal amplification in real time of up to four genes over a wide range of template concentrations. This study shows that multiplexing is a feasible approach applicable to most qRT-PCR assays performed with total RNA, independent of the expression levels of the genes under scrutiny. |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
которые и в ПОДМЕТКИ негодятся АНК-16 чему научит |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
крови или ДОКТОРСКОЙ КОЛБАСЕ нету СМЫСЛА РЕАЛТАЙМИТь Тетрада на 1054 реакции и ВПЕРЕД ! Как работатъ с 384 ПЦР плашками - почитайте в "МЕТОДАХ" на этом сайте |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
Multiplex PCR: rapid DNA cycling in a conventional thermal cycler. * Markoulatos P, * Siafakas N, * Katsorchis T, * Moncany M. Virology Department, Hellenic Pasteur Institute, Athens, Greece. markoulatos@mail.pasteur.fr Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial. Copyright 2003 Wiley-Liss, Inc. PMID: 12784258 [PubMed - indexed for MEDLINE] |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
30 циклов за 11 минут |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
это с КАКОЙ ЦЕЛьЮ - типа если мало - то можно и перелить ? |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection. * Hernandez M, * Rodriguez-Lazaro D, * Esteve T, * Prat S, * Pla M. Dpt de Genetica Molecular, Institut de Biologia Molecular de Barcelona, Centro de Investigacion y Desarrollo-Consejo Superior de Investigaciones Cientificas, Jordi Girona 18-26, 08034, Barcelona, Spain. Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification. |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
1: Biotechniques. 2005 Dec;39(6):885-93. Links High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. * Wang J, * Chuang K, * Ahluwalia M, * Patel S, * Umblas N, * Mirel D, * Higuchi R, * Germer S. Human Genetics Department, Roche Molecular Systems, Alameda, CA 94501, USA. jun.wang@roche.com Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature -shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instrume |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
видно на КРИВОЙ ПЛАВЛЕНИЯ с Евой и СИТО9 на ОДНОКАНАЛьНИКЕ ? |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
- да и ПЦРиш на "обычном" циклере типа Тетрады а на Реалтайме тока Плавишь Как Учит Папа Хигучи ))))))))))))9 |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
1: Clin Chem. 2006 Mar;52(3):494-503. Epub 2006 Jan 19.Click here to read Links Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. * Herrmann MG, * Durtschi JD, * Bromley LK, * Wittwer CT, * Voelkerding KV. Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT, USA. mark.herrmann@aruplab.com BACKGROUND: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated. METHODS: A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a rate of 0.1 degrees C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature and to scan for heterozygotes by curve shape was evaluated. RESULTS: Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 degrees C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by T(m), but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity. CONCLUSION: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis. PMID: 16423901 [PubMed - indexed for MEDLINE] |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
сейчас про патенты 1: Methods Mol Biol. 2006;335:95-114.Click here to read Links Homogeneous detection of nucleic acids using self-quenched polymerase chain reaction primers labeled with a single fluorophore (LUX primers). * Nazarenko I. Research and Development, Digene Corporation, Gaithersburg, MD, USA. Multiplex quantitative polymerase chain reaction (PCR) based on novel design of fluorescent primers is described. Self-quenched fluorogenic primers are labeled with a single fluorophore on a base close to the 3'-end with no quencher required. A tail of 5-7 nucleotides is added to the 5'-end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) are as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Self-quenched primers could be designed manually or by specialized software and could be used for real-time gene quantitation. Targets of 10-107 copies could be detected with precision in PCR using fluorescein-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method could also be used to detect single nucleotide polymorphism by allele-specific PCR. In conclusion, self-quenched primers are an efficient and cost-effective alternative to fluorescence resonance energy transfer-labeled oligonucleotides. |
passer-by IP-штамп: frHUT32Ppt4zQ гость |
Замечания по патентным материям мне кажутся несколько притянутыми за уши, поскольку в Российском и (таком обожаемым некоторыми товарищами) американском патентном законодательствах есть различия, а мы живем в России. Так что ежели кто хочет повозить ялекса "мордой по патенту", указывайте, плз, соответствующие Российские патентные документы ... Извините все за оффтоп... тема все-таки интересная
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Guest IP-штамп: fr45uHVgO7WZ2 гость |
(passer-by @ 09.02.2007 17:04) Товарищ Гуест, Вы, конечно, гений ПЦР-а, но нельзя ли по-медленнее - не все такие умные тут как Вы. А то ваши мощные всплески интеллекта, опережающие возможности стандартной клавиатуры, производят мрачное о вас впечатление - читается с большим трудом и навевает мысли о медицине... (не подумайте, что хочу Вас обидеть, просто прошу подумать и о тех, кто читает Ваши тексты) Замечания по патентным материям мне кажутся несколько притянутыми за уши, поскольку в Российском и (таком обожаемым некоторыми товарищами) американском патентном законодательствах есть различия, а мы живем в России. Так что ежели кто хочет повозить ялекса "мордой по патенту", указывайте, плз, соответствующие Российские патентные документы ... Извините все за оффтоп... тема все-таки интересная вроде различия есть в патентном заквстве- тока мне не оченъ понятна вся эта история с учителем деревенской школы в Перми который обокрал Билла Гейтца |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
если я не путаю "РУ" с Румынией |
Guest IP-штамп: fr45uHVgO7WZ2 гость |
(Guest @ 12.02.2007 18:07) вроде Пелтье цыклер с горячей крышкой Перкин-Елмер запантентовал в России ? если я не путаю "РУ" с Румынией Patent Number WO 98/43740 |
inch5 |
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Van Bok |
(jalex @ 16.06.2003 17:33) Российские RT-PCR и секвенатор есть! (То, о чём так долго говорили большевики.) На выставке "Биотехнология 2003" (Питер, 16-19 июня) приборы можно увидеть в работе и пообщаться с людьми, которые делали железо, оптику, химию, разрабатывали методы, проводили настройку, отладку и ещё много-много всего. |
nigoal123 IP-штамп: frpXPq5TOmUHs гость |
and who it is targeted at. |
basit6666 IP-штамп: frNmR5X46wtQE гость |
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guest: 123 IP-штамп: frJhOCvSv9ICE гость |
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