Comparison between Four Types of Buffers for DNA Agarose Gel Electrophoresis

To detect the genome of viruses (in environmental and clinical samples), we use electrophoresis running buffer after PCR reaction. Also, electrophoresis buffers were used widely to separate any DNA molecule. In this paper, we used four types of previously known electrophoresis buffers to compare which is easy for preparation, simple in structure, low cost and good performance in agarose gel electrophoresis. For this, we used two agarose concentration (1%, 2%) and two types of DNA ladder (100 bp, 1 kb) represent both smaller and larger sizes of molecule for each type of buffers, from the result we found in first level both supper buffer and TAE buffer with good performance and in second level we found bicarbonate buffer also with good performance also. Finally, we found the tang buffer cannot pose any electrophoretic activity on DNA agarose gel electrophoresis.

基于PCR-DGGE技术分析浓香白酒窖泥梭菌多样性

窖泥是浓香型白酒酿制过程中最主要的微生物源,窖泥中微生物的类型、丰度、新陈代谢活动等均对浓香型白酒品质产生很大的影响。为探究窖泥中梭菌微生物的多样性,利用窖泥理化结合聚合酶链式反应-变性梯度凝胶电泳技术对10个窖泥样品中的梭菌群落及变化规律进行研究。结果表明,所选10个窖泥的理化参数均符合优质窖泥指标要求;在微生物层面,窖泥中检测到的梭菌在属水平上有:嗜碱菌属、丁酸弧菌属、梭菌属、喜热菌属、瘤胃梭菌属、粪球菌属、沉淀杆菌属(Sedimentibacter)、钙原杆菌属(Caldicoprobacter)、温带菌(Tepidimicrobium)、梯氏菌(Tissierella)、孢子菌(Sporanaerobacter)、硫酸盐还原菌属、鲁替孢菌属和梭状芽胞杆菌(Clostridiisalibacter)等,这些菌是优质窖泥的重要指示菌,可知窖泥中含有极其丰富的酿酒功能菌。揭示了可能在白酒酿造中起关键作用的梭菌菌群,在分子水平上为研究浓香型白酒提供了理论依据。

芝麻香型高温大曲制曲过程中理化生化与细菌DGGE指纹图谱的探究

采用16S rRNA、PCR-变性梯度凝胶电泳(PCR-DGGE)技术,在芝麻香型高温大曲培曲及前期储存期间进行采样,并分别对高温大曲的各项理化生化指标变化规律及细菌DGGE指纹图谱进行综合分析,揭示了在高温制曲及贮存进程中,各项理化生化指标的变化规律及细菌种类具有多样性及相似性,该探究为指导高温大曲功能微生物筛选应用、优化提升大曲制作工艺提供参考。

Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) Profile of Bacterial Community from Agricultural Soils in Bauchi, North-East Nigeria

The population dynamics of bacterial community was investigated in three Agricultural soils, designated as Loamy sand (A), Peaty coarse (B) and Loamy coarse sand (C) in North-East, Nigeria. The soil chemical properties were characterized to fully understand their nature. Metagenomic approach was used to extract soil DNA using the fast DNA Spin Kit extraction technique. The PCR-electrophoresed DNA bands were excised and subjected to a full scale Denaturing Gradient Gel Electrophoresis (DGGE) analysis. DGGE fingerprinting for the PCR-16S rDNA product revealed a diverse profile of complex population of bacterial community in the study area. The study shows that more bacterial community can be fully investigated using molecular techniques rather than traditional culture method. The implication of the results obtained is discussed.

Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

A New Capillary Electrophoresis Apparatus with Piezoelectric Ceramics High Voltage Source and Amperometric Detector

IntroductionHighperformancecapiliar}'electrophoresis(HPCE)isanewanalyticaltechnology'rapidly'developedinrecentyears.Withtheadvantagesofsmallsample.highsensitivity,highresolution.rapidanalysisandverycheaprunning,ithasbeenappliedinchemistry'.lifescienc...

Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis （2-DE） analysis in kenaf leaf tissues, three extraction methods （trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods） were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects： the pH range of IPG （immobilized pH gradient） stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of （26.40±0.859）%, showed （25.67±1.53） protein bands in one dimension SDS-PAGE gels, and （1 374±54.44） protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips （linear pH gradient of 4-7）, 1.4 mg samples, and 12% SDS-PAGE gels.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Capillary electrophoresis with electrochemiluminescence detection for the analysis of quinolone drugs and pharmacokinetics study

A novel method for the determination of two quinolone drugs norfloxacin （NOR） and levofloxacin （LVX） was described by capillary electrophoresis with electrochemiluminescence detection. The good relationship （r ≥ 0.9991） between peak area and concentration of analytes was established over two orders of magnitude. The limits of detection （LOD, S/N = 3） in standard solution are 4.8 × 10^-7 mol/L for NOR and 6.4 × 10^-7 mol/L for LVX, respectively. The limits of quantitation （LOQ, S/N = 10） in real human urine samples are 1.2 × 10^-6 mol/L for NOR and 1.4 × 10^-6 mol/L for LVX, respectively. The present method was successfully applied to the determination of NOR and LVX in human urine and the studv of oharmacokinetics of NOR.

NEW APPROACH TO MINIMIZE DISPERSION INDUCED BY TURN IN CAPILLARY ELECTROPHORESIS CHANNEL FLOWS

The mechanism of dispersion induced by turn in the capillary electrophoresis channel flows was analyzed firstly. Then the mathematical model of electroosmotic flow is built, and the dispersion of the flow, with different distribution of charge at inner and outer wall in the turns, was simulated numerically using the finite differential method. A new approach of altering the distribution of charge at inner and outer wall in the turns was presented, based on the computational results, to minimize the dispersion induced by turn. Meanwhile, an optimization algorithm to analyze the numerical results and determine the optimal distribution of charge in the turns was also developed. It is found that the dispersion induced by turn in the capillary electrophoresis channel flows could be significantly suppressed by this approach.

Determination of Nicotine in Tobacco by Capillary Electrophoresis with Electrochemical Detection

A sensitive, simple and low-cost method based on capillary electrophoresis（CE） with electrochemical（EC） detection at a carbon fiber microdisk electrode（CFE） was developed for the determination of nicotine. Effects of de- tection potential, concentration and pH value of the phosphate buffer, and injection time as well as separation voltage were investigated. Under the optimized conditions： a detection potential of 1.20 V, 40 rnmol/L phosphate buffer（pH 2.0）, a sample injection time of 10 s at 10 kV and a separation voltage of 16 kV, the linear range obtained was from 5.0×10^-7 mol/L to 1.0×10^-4 mol/L with a correlation coefficient of 0.9989 and the limit of detection（LOD, S/N=3） obtained was 5.0×10^-8 mol/L. The method was also used to determine the nicotine in cigarettes. Nicotine amount ranged from 0.211 mg/g to 0.583 mg/g in the pipe tobacco of seven brands of cigarette and the amount in one ciga- rette varied from 0.136 mg/cigarette to 0.428 mg/cigarette.

Determination of Magnolol and Honokiol by Non-aqueous Capillary Electrophoresis

Two active principles in traditional Chinese medicine Magnolia officinalis, magnolol and honokiol, were successfully separated by means of nonaqueous capillary electrophoresis. The effect of the composition of a nonaqueous buffer on column efficiency and resolution, and the effect of acid additives on peak shapes were researched. The separation was conducted with a running buffer in a mixture of methanol/aeetonitrile/formamide （ volume ratio ： 1 ： 2 ： 2 ）, in which the concentrations of Tris, acetic acid, and water were 60 retool/L, 0. 04 mmol/L and 5% （ volume fration）, respectively, and the pH^＊ （apperent pH） of the running buffer was 8.96. Magnolol and honokiol were separated on baseline within 20 min. The relative standard deviation of the analytes＇ concentrations in the sample is 1.32% for magnolol and 1.60% for honokiol, and the recoveries of the spiked sample are 98.4% for magnolol and 98. 0% for honokiol, respectively.

Studies on Human Immunoglobulin G from GBS Patient (Ⅲ)-The Determination of Molecular Weight of Human Immunoglobulin G by Capillary SDS Gel Electrophoresis

Guillain-Barre syndrome (GBS) is considered to be an autoimmune disorder of peripheralnervous system. In this paper. capillary SDS gel electrophoresis was performed on neutral coatedfused-silica capillary to determine the molecular weight of purified IgG samples from GBS patient.

Determination of urinary 8-hydroxy-2′-deoxyguanosine by capillary electrophoresis with molecularly imprinted monolith in-tube solid phase microextraction

Urinary 8-hydroxy-2 -deoxyguanosine(8-OHdG) is an excellent marker of oxidative DNA damage.In this study,employing guanosine as dummy template a novel molecularly imprinted(MIP) monolithic capillary column had been synthesized,and that was used as medium of in-tube solid phase microextraction(SPME).Coupled with capillary electrophoresis-electrochemical detection(CE-ECD),the system of extraction and detection of 8-OHdG in urinary sample had been developed.Because of its greater phase ratio combined with conv...

A novel method for Co(Ⅱ) and Cu(Ⅱ) analysis by capillary electrophoresis with chemiluminescence detection

Based on the fact that some metal ions can catalyze the chemiluminescence(CL)reaction of luminol with K_3Fe(CN)_6,a novel capillary electrophoresis CL method was developed for the determination of Co(Ⅱ)and Cu(Ⅱ).The separation was carried out with a 10 mmol/L sodium acetate solution containing 0.8 mmol/L luminol and 2.0 mmol/Lα-HIBA(adjusted to pH 4.8 by HAc solution).The post-capillary reagent was 2.0 mmol/L K_3Fe(CN)_6 which was adjusted to pH 13.0 by NaOH solution.Under the optimum conditions,the detection limits(S/N=3)for Co(Ⅱ)and Cu(Ⅱ)were 7.5×10^(-11)mol/L and 7.5×10^(-9)mol/L,with the linear range of 7.5×10^(-9)mol/L to 1.0×10^(-6)mol/L and 7.5×10^(-8)mol/L to 5.0×10^(-5)mol/L, respectively.

Comparative Proteomic Analysis of B. henselae Houston and B. henselae Marseille by Two-dimensional Gel Electrophoresis

To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. Method Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis. Results Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 μg/μL and 2.200 μg/μL respectively. Sample protein of 40 μg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae. Conclusion The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.

Synthesis and Characterisation of Water-Soluble Carboxymethyl-Cyclodextrin Polymer as Capillary Electrophoresis Chiral Selector

The water-soluble carboxymethyl-cyclodextrin polymer (CM-CD polymer) was synthesized and used as capillary electrophoresis chiral selector. Verrapamil and thiopentorusodium were well separated using CM-CD polymer as chiral selector.

Study on Rhizoma Chuanxiong based on capillary electrophoresis with amperometric detection

A high-performance capillary electrophoresis with amperometric detection(CE-AD) method has been developed for the analysis of seven bioactive ingredients,namely ferulic acid(FA),vanillin,vanillic acid,p-hydroxybenzoic acid,caffeic acid,gallic acid and protocatechuic acid,in Rhizoma Chuanxiong.The effects of several factors such as the acidity and concentration of running buffer,the separation voltage,the applied potential to working electrode and the injection time were investigated.Under the optimum condit...