Development and verification of chemical chromogenic method for determination of residual cetyltrimethyl ammonium bromide content in ACYW135 meningococcal polysaccharide stock solution / 中国生物制品学杂志

@#Objective To develop and verify a chemical choromogenic method for the determination of residual cetyltrimethylammonium bromide(CTAB)content in ACYW135 meningococcal polysaccharide stock solution. Methods A chemical chromogenic method was developed for the determination of residual CTAB content by using titan yellow as chromogenic reagent,and verified for the linear range,intermediate precision and accuracy. ACYW135 meningococcal polysaccharide stock solution were determined for residual CTAB content by the developed method. Results The CTAB reference at concentrations of 4. 0～10. 0 μg/mL showed good linear relationship to A_(500),with a R~2 value of more than 0. 990. The recovery rates of CTAB standard at concentrations of 5. 0,7. 0,and 9. 0 μg/mL were all within 95% ～ 110% in six repeated tests,with the CV values of determination results of less than 10%. All the residual CTAB contents in four batches of meningococcal polysaccharide stock solutions were less than 8. 0 μg/mL. Conclusion The chemical chromogenic method showed good linearity,intermediate precision and accuracy,which might be used for the determination of residual CTAB content in ACYW135 meningococcal polysaccharide stock solution.

Evaluation of uncertainty in determination of cetrimonium bromide residue in polysaccharide vaccines by ultra performance liquid chromatography-mass spectrometry / 中国生物制品学杂志

@#Objective To determine the cetrimonium bromide(CTAB)residue in polysaccharide vaccines using ultra performance liquid chromatography-mass spectrometry(UPLC/MS-MS),and analyze and evaluate the uncertainty of the determination results.Methods By establishing a mathematical model,the sources and values of uncertainty introduced in the measurement process were analyzed,the uncertainty components of each influencing factor were calculated,and the standard uncertainty and expanded uncertainty were synthesized to form an uncertainty report.Results At 95% confidence interval,the expanded uncertainty was 0. 002 8 mg/kg. The determination result of CTAB residue in polysaccharide vaccine was reported as(1. 000 6 ± 0. 002 8)mg/kg(k = 2,confidence interval p = 95%).Conclusion The main factors affecting the accuracy of determination results are the preparation of standard solution and the introduction of recovery rate,which should be focused on and controlled in the experiment process to make the detection results more reliable.

Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of ß-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA / Preservação e maceração de tecido de folíolos de açaí da Amazônia para obtenção de DNA genômico

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µ L (7 days in transport buffer) to 702.00 ng/µ L (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µ L (30 days in silica gel) to 2,850.00 ng/µ L (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

O objetivo deste estudo foi testar a eficiência de métodos de preservação e maceração de tecidos de folíolos de Euterpe precatoria para obter DNA genômico para estudos moleculares. Os folíolos de E. precatoria foram coletados no campo experimental da Embrapa Acre, Brasil. O estudo foi conduzido em delineamento inteiramente casualizado com 10 repetições, em esquema fatorial 12 × 2, com 12 tratamentos de armazenamento (fresco; liofilizado 3 dias; geladeira 3, 5, e 7; sílica gel 7, 10, 20 e 30 dias e tampão de transporte 3, 5 e 7 dias) e dois tipos de maceração do tecido foliar (nitrogênio líquido e TissueLyser®). Para a variável concentração de DNA houve diferença estatística entre os tipos maceração e de armazenamento. O macerador TissueLyser® apresentou maiores concentrações de DNA quando comparado ao nitrogênio líquido.Para os tipos de armazenamento verificou-se formação de cinco grupos quando macerados TissueLyser® e dois grupos quando macerados com nitrogênio líquido. As concentrações de DNA variaram de 285,00 ng/µ L (7 dias em tampão de transporte) a 702,00 ng/µ L (30 dias em sílica gel) quando maceradas com nitrogênio líquido. Quando maceradas com macerador TissueLyser® variaram de 572,73 ng/µ L (30 dias em sílica gel) a 2.850,00 ng/µ L (3 dias em liofilizador). A pureza do DNA (A260/A280 nm) variou de 1,30 a 1,70 quando os folíolos foram macerados com nitrogênio líquido e de 1,30 a 1,90 quando macerados em macerador TissueLyser®. Apesar das variações na conservação e concentração dos tecidos foliares, todos os tratamentos foram eficazes para o isolamento do DNA e amplificaram regiões de marcadores microssatélites. Concluiu-se que folíolos de E. precatoria armazenados em liofilizador e macerados com macerador automático resultaram em DNA satisfatório para estudos moleculares.

Comparison of three different methods for isolating RNA from Oncomela-nia hupensis / 中国血吸虫病防治杂志

Objective To compare the effects of three kinds of Oncomelania hupensis RNA extraction methods,namely a modified SDS method,TRIzol reagent method,and CTAB method,so as to obtain an economical and efficient method for RNA extraction from O. hupensis. Methods The modified SDS method,TRIzol reagent method and CTAB method were applied to ex-tract the RNA from O. hupensis. A nucleic acid protein analyzer was used to measure the concentration and purity of RNA. The yields were calculated by the concentration of the products. The purity was indicated by A260/A280 and A260/A230. The quality of RNA was inspected by 1% agarose gel electrophoresis. The β-acting gene was selected as the target gene for RT-PCR analysis. Re-sults The RNA yields obtained by using the three kinds of extraction methods were significantly different(F = 16895.85,P <0.01)according to the analysis of variance. The LSD test showed that the yields obtained by using the modified SDS method were the highest,and those obtained by the CTAB method were the lowest. The purity of RNA extracted by the CTAB method was su-perior to that by the other two methods,and the A260/A280 and A260/A230 ratios of the CTAB method were in the range from 1.8-2.0 and 2.0-2.2. The A260/A230 ratios of the other two methods were both lower than 2.0. The RNA extracted by the modified SDS meth-od had the better integrity. The electrophoresis results showed that the 28S rRNA band,18S rRNA band and 5S rRNA band were clear,and there was no obvious smear between each band. The RNA obtained by the TRIzol reagent method had no 28S rRNA band,and that obtained by the CTAB method had no 28S rRNA and 5S rRNA bands. The β-acting gene of the RNA ex-tracted by all the three methods could be amplified by RT-PCR. The costs and time-consuming of the modified SDS method were less than those of the other two methods. Conclusion The modified SDS method is an economic and efficient method,and it is suitable for extracting the RNA of O. hupensis,especially for large sample preparation.

Optimización y modificación del método para la detección de ramnolípidos

El uso de biosurfactantes en biorremediación facilita y acelera la degradación microbiana de hidrocarburos. El método del agar CTAB/MB creado por Siegmund y Wagner para el screening de cepas productoras de ramnolípidos (RL), ha sido ampliamente utilizado sin sufrir mejoras significativas en más de 20 años. Con el fin de optimizar la técnica como método cuantitativo, se hicieron placas con agar CTAB/MB y se probaron diferentes variables, tales como tiempo de incubación, refrigeración, concentración de CTAB, presencia de azul de metileno, diámetro de los pozos y volumen del inóculo. Adicionalmente, se desarrolló un nuevo método para detectar el RL de los halos mediante precipitación con HCl, lo cual permite la observación de un nuevo patrón de halos más fáciles de observar y medir. Esta investigación reafirma que este método no es del todo apropiado para un análisis cuantitativo fino, debido a la dificultad de correlacionar de forma precisa la concentración de RL y el área de los halos formados. La difusión del RL no parece tener un comportamiento simple y existen numerosos factores que afectan la velocidad de migración del RL.

Use of biosurfactants in bioremediation, facilitates and accelerates microbial degradation of hydrocarbons. CTAB/MB agar method created by Siegmund & Wagner for screening of rhamnolipids (RL) producing strains, has been widely used but has not improved significantly for more than 20 years. To optimize the technique as a quantitative method, CTAB/MB agar plates were made and different variables were tested, like incubation time, cooling, CTAB concentration, methylene blue presence, wells diameter and inocula volume. Furthermore, a new method for RL detection within halos was developed: precipitation of RL with HCl, allows the formation a new halos pattern, easier to observe and to measure. This research reaffirm that this method is not totally suitable for a fine quantitative analysis, because of the difficulty to accurately correlate RL concentration and the area of the halos. RL diffusion does not seem to have a simple behavior and there are a lot of factors that affect RL migration rate.

Optimization of soybean dna extraction under different storage and development periods / Otimização de extração de dna de soja sob diferentes períodos de armazenamento e desenvolvimento

DNA extraction of plants with high quality is very important to researches in molecular biology. Several extraction protocols have been used to obtain soybean DNA; however, there is a lack of papers about extraction protocols optimization and the best developmental stage of the plant to collect them. Therefore, the main purpose of the study was to extract high quantity and quality of DNA from fresh or frozen soybean samples, using different protocols. Moreover, we analyzed the best developmental stage of the plant to do the extraction. Fresh leaves or leaves kept for two years in the ultra-freezer were submitted to the DNA extraction protocols: Haberer et al., 1996 (modified); second modification from Haberer et al., 1996; Murray & Thompson, 1980 (modified) e Doyle & Doyle, 1990 (modified). Modified protocol of Doyle & Doyle was used to value the best stage to collect the leaves to do the DNA extraction. The samples were collected in the stages of development VC, V1, V2, V3, V4 and R5. The experiments were conducted in completely randomized design with 10 samples per treatment. The data underwent variance analysis and the averages were compared by the Tukey test (p<0.05). Through Doyle & Doyle, 1990 and Haberer et al., 1996 modified protocols, for both fresh and frozen samples, it was possible to obtain a higher total DNA concentration if compared to the other tested protocols. However, the quality of DNAs extracted by the protocol Doyle & Doyle, 1990 (modified) was superior, due to a minor molecular degradation. Besides that, the extractions made with these protocols have shown to be more efficient using frozen leaves' tissue. Higher DNA concentrations were obtained analyzing VC samples; however, there were no statistical differences between the stages VC, V2 and V3. It is suggested thereby to use modified of Doyle & Doyle for DNA extraction from soybean leaves in V2 and V3 stages of development from frozen samples, providing the collect of a large number of samples and its storage until the analysis.

A extração de DNA de plantas com alta qualidade é de suma importância para pesquisas em biologia molecular. Diversos protocolos de extração vêm sendo utilizados para a obtenção de DNA de soja; contudo, há uma carência de trabalhos de otimização de protocolos de extração e de escolha do melhor estádio de desenvolvimento da planta para a coleta. Desta forma, o objetivo do estudo foi extrair DNA com alta quantidade e qualidade a partir de amostras frescas ou congeladas de soja, utilizando diferentes protocolos de extração. Além disso, foi analisado o melhor estádio de desenvolvimento da planta para a extração. Folhas frescas e armazenadas por cerca de dois anos em ultrafreezer foram submetidas aos protocolos de extração de DNA: Haberer et al., 1996 (modificado); segunda modificação de Haberer et al., 1996; Murray & Thompson, 1980 (modificado) e Doyle & Doyle, 1990 (modificado). Para a avaliação do melhor estádio de coleta das folhas para a extração de DNA foi utilizado o protocolo de Doyle & Doyle modificado. As coletas de amostras foram realizadas nos estádios de desenvolvimento VC, V1, V2, V3, V4 e R5. Os experimentos foram conduzidos em delineamento inteiramente casualizado com 10 amostras por tratamento. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Turkey (p<0,05). Através dos protocolos modificados de Doyle & Doyle, 1990 e Haberer et al., 1996, tanto para amostras frescas como para congeladas, foi possível obter uma maior concentração de DNA total se comparado aos demais protocolos testados. Porém, a qualidade dos DNAs extraídos pelo protocolo Doyle & Doyle, 1990 (modificado) foi superior, devido a menor degradação da molécula. Além disso, as extrações efetuadas com estes protocolos se mostraram mais eficientes quando foram utilizados tecidos foliares congelados. Maiores concentrações de DNA foram obtidas quando amostras em VC foram analisadas; porém, não houve diferença estatística entre os estádios VC, V2 e V3. Assim, sugere-se a utilização do protocolo modificado de Doyle & Doyle para extração de DNA de folhas de soja nos estádios de desenvolvimento V2 e V3 a partir de amostras congeladas, viabilizando a coleta de um grande número de amostras e o seu armazenamento até a análise.

Comparison of Different Extraction Methods of Genomic DNA of Cordia Dicholoma Seeds / 中国中医药信息杂志

Objective To extract high quality genomic DNA of Cordia dicholoma seeds by using different methods;To provide references for researches on genomic DNA of Cordia dicholoma seeds. Methods Genomic DNA of Cordia dicholoma seeds was extracted through improved CTAB method and improved SDS method. Purity and concentration of obtained DNA were detected by spectrophotometry and agarose gel electrophoresis. Results The results of spectrophotometry showed that the purity of genomic DNA obtained through improved CTAB method was better than improved SDS method. Genomic DNA extracted through improved CTAB method was without protein and RNA pollution. The results of agarose gel electrophoresis showed that electrophoresis of genomic DNA obtained through the two methods both had the main belt. However, genomic DNA extracted through improved SDS method degraded more than improved CTAB method. Conclusion Improved CTAB method can obtain relatively high quality genomic DNA of Cordia dicholoma seeds.

Comparación de métodos de extracción de RNA para la detección por RT-PCR del Potato yellow vein virus (PYVV) en diferentes órganos de Solanum tuberosum Grupo Phureja / Comparison of RNA isolation methods for Potato yellow vein virus (PYVV) detection by RT-PCR in different organs of Solanum tuberosum Group Phureja

Potato yellow vein virus (PYVV, Crinivirus/Closteroviridae), contiene genoma tripartita de ssRNA(+), se limita al floema y causa pérdidas en la producción. Es un virus re-emergente y cuarentenario en Europa y Estados Unidos. La detección se ha basado en RT-PCR, NASH y RT-PCR en tiempo real (RT-qPCR) en muestras de foliolo y tubérculo. Se desconoce como es la distribución del virus en plantas infectadas lo que hace necesario establecer un método de extracción de RNA, que sea eficiente en la obtención de material a partir de diferentes órganos. El objetivo de este trabajo fue comparar tres métodos de extracción de RNA: uno basado en Tioisocianato de guanidina (Trizol® (Invitrogen)), uno que utiliza bromuro de hexadecil trimetil amonio (CTAB) y el método fenol/cloroformo seguido por purificación con columnas de Sephadex; para detectar el virus por RT-PCR en: foliolo, peciolo, pedúnculo floral, pétalos, tallo aéreo y subterráneo, antera y brotes de tubérculo; de plantas infectadas. Los métodos de extracción fueron evaluados en términos de la integridad (relación de la intensidad de las bandas de las subunidades ribosomales 28S/18S), calidad (relación de las lecturas espectrofométricas 260/280nm) y rendimiento de los extractos. PYVV se detectó por RT-PCR en todos los órganos analizados por los tres métodos de extracción. No se presentaron diferencias estadísticamente significativas entre los tres métodos de extracción; sin embargo, Trizol® (Invitrogen) presentó mayor rendimiento, calidad e integridad; además, permitió la detección del virus por RT-PCR en todos los órganos evaluados.

Potato yellow vein virus (PYVV, Crinivirus/Closteroviridae), contains a tripartite genome with ssRNA(+), is phloem limited and can affect yield reduction. PYVV is a re-emergent virus and is a quarantine pathogen in Europe and United States. The detection has been based in RT-PCR, NASH and real time RT-PCR (RT-qPCR) in leaflets and tuber shoots samples. It is not known how is the distribution of PYVV within infected plants, for that is necessary to establish an efficient RNA isolation method for obtaining material from different organs. The objective of the present work was to evaluate three RNA isolation methods: a Trizol® (Invitrogen) based method, a method using hexadecyltrimethylammonium bromide (CTAB) and the phenol - chloroform method followed by Sephadex columns purification; for virus detection using RT-PCR in: leaflets, petiole, peduncle, petals, stem, aerial and subterranean roots, anther and shoot tuber; of infected plants. The isolation methods were tested in terms of integrity (ratio of band intensity of 28S and 18S ribosomal subunits), quality (absorbance 260/280 ratio) and total yield. PYVV was detected by RT-PCR in all organs analyzed by the three isolation methods. There were no significant differences found among the three isolation methods, although, Trizol® (Invitrogen) presented high yield, quality and integrity, furthermore Trizol® (Invitrogen) allowed the virus detection using RT-PCR in all analyzed organs.

DESARROLLO DE MÉTODOS DE ANÁLISIS POR INYECCIÓN EN FLUJO A PARTIR DE PROCEDIMIENTOS CLÁSICOS: Determinación de Pb en agua / DEVELOPMENT OF FLOW INJECTION ANALYSIS METHODS BY CLASSICAL PROCEDURES: Determination of Pb in water / DESENVOLVIMENTO DE MÉTODOS DE ANÁLISE POR INJEÇÃO EM FLUXO A PARTIR DE PROCEDIMENTOS CLÁSSICOS: determinação de Pb em água

El análisis químico día a día se acerca más a la automatización, buscando satisfacer las necesidades actuales de resultados rápidos y confiables. Los sistemas de análisis en flujo (FIA - Flow Injection Analysis) son una de las formas de aproximarse a la automatización. En este artículo se presentan los pasos necesarios para implementar una metodología FIA, para la determinación de Pb(II) en agua, partiendo de la revisión de los procedimientos clásicos y describiendo detalladamente los pasos necesarios para implementar la técnica de análisis en flujo. El trabajo produjo un método de análisis de Pb en agua que usa ditizona disuelta en isopropanol (agente cromogénico), en presencia de bromuro de cetiltrimetil amonio (CTAB), para solubilizar en agua el complejo, cuyas características más sobresalientes fueron: volumen de inyección de muestra de 81,7 µL, velocidad de flujo de 8,0 mL/min, tiempo de toma de espectros 1,4 s e intervalo lineal de 1,0 a 40 mg L-1.

Chemical analysis has evolved towards automation to satisfy the current requirements: fast analysis and certainty in the results. Flow injection analysis (FIA) is a way to reach automation. This work presents the necessary steps to obtain an optimized FIA methodology for the determination of Pb(II) in water by classic methods. The result was a FIA method to determinate Pb with dithizone (chromogenic agent) dissolved in iso-propyl alcohol, using cethyltrimethylammonium bromide (CTAB) to solubilize the complex. The main characteristics of the method were: injection sample volume 81.7 µL, flow 8.0 mL/min, spectra acquisition time 1.4 s and linear range 1 to 40 mg L-1.

Cada dia, a análise química é mais cerca da automatização com o fim de satisfazer as necessidades atuais de resultados rápidos e confiáveis. Os sistemas de análise em fluxo (FIA - Flow Inyection Analysis) são uma das formas de aproximação à automatização. Este artigo apresenta os passos necessários para implementar uma metodologia FIA para a determinação de Pb(II) em água, partindo da revisão dos procedimentos clássicos e descrevendo detalhadamente os passos necessários para implementar a técnica de análise em fluxo. Os resultado são um método de análise de Pb em água que usa ditizona dissolvida em isopropanol (agente cromogénico) na presença de bromuro de cetiltrimetil amônio (CTAB), usado para solubilizar o complexo em água. As características principais do método foram: volume de injeção de amostra de 81,7 µL, velocidade de fluxo de 8,0 mL min-1, tempo de aquisição de espectros de 1,4 s e intervalo linear de 0.9 a 40 mg L-1.

Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB), for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

Detergent stable, halotolerant -amylase from bacillus aquimaris vitp4 exhibits reversible unfolding.

Halotolerant Bacillus aquimaris VITP4, isolated from Kumta coast (India), was used to produce extracellular α-amylase. The production was found to be maximal after 24 h of growth at pH 8.0 and 40 oC. Optimal activity of the purified enzyme was in the pH range of 7.5 – 9.5 at 40 oC. The enzyme was found to retain more than 60% of the initial activity even at NaCl concentration of 3.5 M, indicating that it is halotolerant. Calcium ion (0.01 mM) and CTAB (10 mM) enhanced the activity whereas EDTA decreased the enzymatic activity. Thermal inactivation kinetics suggested that the enzyme exhibits reversible unfolding even at high temperature (till 90 oC) and the t1/2 at 90 oC was found to be 43.5 min. These results suggests that the α-amylase from Bacillus aquimaris strain VITP4 is halotolerant, metal ion dependent enzyme which is stable in the presence of cationic detergent and moderate temperature conditions.

Methodological variations in the isolation of genomic DNA from Streptococcus bacteria

In this work, genomic DNA of Streptococcus pyogenes, S. mutans and S. sobrinus was isolated using two methods: either using the detergent cetyltrimethylammonium bromide (CTAB) at 65ºC; or by applying ultrasound to a mixture of silica and celite in CTAB. The composite method that used ultrasound was the more efficient, allowing the straightforward extraction of genomic DNA from Gram-positive bacteria with good quality and reproducibility.

O gênero Streptococcus encontra-se amplamente distribuído na natureza e algumas espécies constituem a microbiota humana da cavidade bucal, como Streptococcus pyogenes, que pode estar associado a importantes doenças humanas, Streptococcus mutans e Streptococcus sobrinus, relacionados à cárie dental. O DNA genômico destas três espécies foi isolado utilizando-se dois métodos, o primeiro utilizando o detergente brometo de cetiltrimetilamônio (CTAB) à 65ºC e outro associando ultra-som a uma mistura de sílica e celite em CTAB. O método que possibilitou a extração do DNA genômico das bactérias Gram positivas, com qualidade, boa reprodutibilidade fácil execução foi aquele que utilizou ultra-som associado à sílica e celite em CTAB.

Isolation of genomic DNA from medicinal plants without liquid nitrogen.

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the λ260/λ280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.

Influencing Factors for Genomic DNA Extraction of Paeonia Suffruticosa / 中国药房

OBJECTIVE:To study the influencing factors for the extraction of the genomic DNA from Paeonia Suffruticosa.METHODS:Taking Paeonia Suffruticosa(root bark of Chinese medicinal herb) as material to investigate the influencing factors including concentrations of the NaCl and beta-mercaptoethanol,temperature and time of water bath,RNaseA,PCR(polymerase chain reaction) system etc in the buffer solution on the basis of modified CTAB method.RESULTS:The DNA obtained by modified CTAB method was pure,integrated,with the value of A260/A280 ranged from 1.8 to 2.0,the ampl-ified bands of PCR were clear and bright,which lay a solid foundation for the following molecular biology experiments.CONCLUSION:The modified CTAB method is economical,rapid and efficient,and it can be served as an extraction of genomic DNA from root bark Chinese medicinal herb as well as a theoretical basis for full scale production.

Purification of Kallikrein Using Reverse Micellar Extraction / 中国生物工程杂志

The purification of kallikrein using CTAB/hexanol/octane reverse micelles extraction has been studied and optimized,under various aqueous pH values, ionic strength and species, CTAB concentration and co-surfactant concentration. The result shows that the extraction efficiency approaches 100%, and the activity recovery is more than 80%, the commercial enzyme specific activity is increased by 1.97 times and the crude enzyme activity is increased by 7.15 times, which from 31U/mg to 219U/mg,under the conditions of[CTAB]=0.02 mol/L, hexanol/octane (V/V)=1∶5, pH=9.0 and[KBr]=0.1 mol/L in forward extraction, pH=7.0 ,[KBr]=1.5 mol/L and 15% ethanol(V/V) in backward extraction. The result of purified kallikrein is examined by the SDS-PAGE analysis. Reverse micelles extraction is a potential technique for the application in the downstream biotechnological processes.

A Modified Method for Isolating DNA from Fungus / 微生物学通报

Genomic DNA of two fungi Thamnidium elegans and Umbelopsis isabellina were extracted with an amended Cetyltrimethyl Ammonium Bromide (CTAB) method. This modified method uses repeated freezing in liquid nitrogen and thawing with combination of shocking with glass beads to replace of the tra- ditional method. Quality and concentration of DNA extracted by the modified methodwere tested. Compared with the traditional method, higher yield and purity of genomic DNA were obtained with less amount ofmy- celium. The result indicted that this is a simple and highly efficient method, which is suitable to treat many samples at one time and for basic molecular experiments, such as restriction endonuclease reaction and PCR.