• Title/Summary/Keyword: ethidium bromide

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Improved Detection of ${\gamma}-Irradiated$ Vibrio vulnificus after Heat and Cold Shock Treatment by Using Ethidium Monoazide Real-time PCR

  • Lee, Jung-Lim;Levin, Robert E.
    • Food Science and Biotechnology
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    • v.18 no.3
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    • pp.788-792
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    • 2009
  • Gamma $({\gamma})-irradiation$ can be used to control pathogens such as Vibrio vulnificus in seafood. The effects of irradiation on microbial cell populations (%) have been studied in order to develop detection methods for irradiated foods. The method used in this study was ethidium bromide monoazide (EMA) real-time polymerase chain reaction (PCR), using V. vulnificus specific primer, EMA, and $SYBR^{(R)}$ Green to discriminate between ${\gamma}-irradiated$ and non-irradiated cells. Confocal microscope examination showed that ${\gamma}-irradiation$ damaged portions of the cell membrane, allowing EMA to penetrate cells of irradidated V. vulnificus. ${\gamma}-Irradiation$ at 1.08 KGy resulted in log reduction ($-1.15{\pm}0.13$ log reduction) in genomic targets derived from EMA real-time PCR. The combination cold/heat shock resulted in the highest ($-1.74{\pm}0.1$ log reduction) discrimination of dead irradiated V. vulnificus by EMA real-time PCR.

Analysis of Double-Stranded DNA Fragments by Capillary Electrophoresis Using Entangle Polymer Solutions in Uncoated Fused Silica Capillary Columns

  • Lee, Jong-Jin;Lee, Kong-Joo
    • BMB Reports
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    • v.31 no.4
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    • pp.384-390
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    • 1998
  • DNA fragments (51-587 bp) were separated by capillary electrophoresis using entangled polymer, hydroxyethylcellulose, in uncoated fused silica capillary columns. The factors affecting the separation of DNA fragments with hydroxyethylcellulose media were evaluated, i.e., the concentration of buffer and entangled polymer, effects of additives (methanol, ethidium bromide, EDTA), temperature, and injection methods. Maximum performance was obtained by adding 5% methanol in 0.5% hydroxyethylcellulose solution at $30^{\circ}C$. Addition of methanol in polymer media increased the resolution of small size DNA fragments (< 100 bp). On the other hand, addition of ethidium bromide and EDTA, which are commonly used in conventional DNA separation, reduced the resolution of DNA fragments in the polymer solution. It turns out that the separation behavior of DNA in entangled polymer is more sensitive to the running condition compared to that in polyacrylamide gel-filled capillary, but the reproducibility of DNA separation in entangled polymer is reliable.

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Fluorescence Enhancement of Ethidium Bromide by DNA Bases and Nucleosides

  • Pyun, Chong-Hong;Park, Su-Moon
    • Bulletin of the Korean Chemical Society
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    • v.10 no.2
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    • pp.142-147
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    • 1989
  • Fluorescence enhancements of ethidium bromide (EB) by solution species of low molecular weights such as DNA base molecules and nucleosides in water are reported. The degree of enhancements was determined by intensity as well as lifetime measurements for EB fluorescence. Experiments including solvent effects on absorbance and fluorescence spectra of EB, effects of protonation on the EB absorbance spectrum, and determination of equilibrium constants for EB-DNA bases have been performed to help explain the fluorescence enhancement. The results suggest that the excited state stabilization in the hydrophobic environment, the loss of torsional/vibrational energy of amino groups, and the change in the electronic transition characteristics are all responsible for the fluorescence enhancement.

Host Construction by Curing the Octopine Type Ti and Cryptic Plasmids in Agrobacterium tumefaciens KU12 (Agrobacterium tumefaciens KU12로부터 Octopine형 Ti 및 잠재 플라스미드의 제거에 의한 숙주 개발)

  • Ha, Un-Hwan;Lee, Yong-Woog;Moon, Hye-Yeon;Sim, Woong-Seop
    • Korean Journal of Microbiology
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    • v.32 no.1
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    • pp.53-59
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    • 1994
  • Agrobacterium tumefaciens KU12 contains pTiKU12 (240kb) of the octopine type Ti plamsid and pTi12 (45 kb) of the cryptic plasmid. To make the avirulent A. tumefaciens, the octopine type Ti plasmid, pTiKU12, was cured with elevated temperature (37${\circ}C$) and ethidium bromide (EtBr), respectively. Also the cryptic plasmid, pTi12, was cured by the introduction of recombinant plasmid, pYWXP, made by pTi12 replication origin and pUC19. pYWXP was cured by elevated temperature (37${\circ}C$) and EtBr simultaneously.

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Fluorescence Anisotropy Study on the Effect of Phellodendri Cortex's Berberine on Regulation of the Function of DNA (황백(黃柏)의 berberine이 DNA의 기능조절에 미치는 영향에 관한 형광이방성 연구)

  • Lee, Seong Kyung;Han, Hyo Sang;Huh, Sung Ho
    • The Korea Journal of Herbology
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    • v.33 no.5
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    • pp.105-110
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    • 2018
  • Objectives : We tried to observe the fluorescence anisotropy and intensity of ethidium ion in the intercalating binding interaction between DNA and ethidium ions in the presence of berberine, and then tried to explain the effect of berberine on the intercalating interaction of ethidium ion with DNA. Methods : DNA(calf thymus DNA), berberine and ethidium bromide(EtBr) were purchased from Sigma-Aldrich Co. Proper amount of each compound was dissolved in 20 mM sodium phosphate buffer(pH 7.0) containing 100 mM of NaCl to prepare stock solutions. Collections of the fluorescence anisotropy and intensity data were performed on JASCO FP-8300 spectrofluorometer equipped with a polarizer and a Peltier temperature controller. The excitation of ethidium ion was done at 550 nm and the emission data were collected at 600 nm. For Stern-Volmer plot, the fluorescence data were collected at $18^{\circ}C$ and $30^{\circ}C$. Results : According to the results of this research, the weak competitive binding pattern between ethidium ion and berberine appeared in binding with DNA at low ratio of DNA to ethidium ion. But at high ratio of DNA to ethidium ion, this weak competition disappeared. Instead, berberine might bind to DNA by intercalating way. In other words, berberine could de-intercalate ethidium ion from DNA at low concentration of DNA relative to ethidium ion, but could not at high concentration of DNA relative to ethidium ion. In addition, the mechanism of fluorescence quenching of ethidium ion could also proceed differently, depending on the ratio of the amount of DNA to that of ethidium ion. Conclusions : The effect of berberine on the DNA-ethidium ion intercalating interaction could work differently, depending on the relative ratio of the amount of DNA to that of ethidium ion. This study also showed that fluorescence anisotropy analysis is very useful method to obtain detailed information for investigation of the complex binding interactions. In order to fully understand the mechanism of action of the pharmacological effect by berberine, studies on the effect of berberine on the action of proteins such as various enzymes closely related to berberine-induced medicinal effects should be continued.

A Drug Efflux Pump for Cationic Drugs including Disinfectants in Bacillus subtilis (양이온약제내성을 유도하는 Bacillus subtilis의 Drug Efflux Pump)

  • Yong Joon Chung
    • Microbiology and Biotechnology Letters
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    • v.31 no.3
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    • pp.230-234
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    • 2003
  • The Bacillus subtilis YvaE protein, the small multidrug resistance (SMR) family (TC #2.A. 7.1), is shown to catalyze efflux of multiple cationic drugs including many disinfectants, when it was cloned and expressed in Escherichia coli. When the yvaD gene was coexpressed with yvaE gene, the yvaD protein, encoded within a single operon with the yvaE gene, is shown to counteract the action ofYvaE. By ethidium efflux analysis, the cells harvoring a vector with yvaE gene showed a rapid ethidium efflux, compared with the control cells. These results clearly suggest that YvaE mediates drug export from the cell cytoplasm.

Plasmid-Determined Cadmium Resistance in Cocobacilli Strain B-17 Isolated from Soil. (토양에서 분리된 Cocobacilli B-17균의 Plasmid가 결정하는 Cadmium내성)

  • 방병호
    • The Korean Journal of Food And Nutrition
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    • v.1 no.2
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    • pp.64-67
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    • 1988
  • Cadmium resistant cocobacillus B-17 from soil was tolerated up to 1600ug/ml of cadmium at agar plate and the strain B-17 was able to grow at 600ug/ml of cadmium at liquid medium after the lag phase being prolonged with lengthening culture time. Optimal pH of the strain was shown at pH7.0. The elimination frequency of cadmium resistance by 10ug/ml of acriflavin was 28%, and by 20ug/ml of ethidium bromide was 47%, respectively.

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Transfer of RNA from Methylmercury-agarose Gel to Nitrocellulose Membrane (메칠머큐리젤에 분리된 리보핵산의 니트로셀루로스막으로 이동)

  • 안정선
    • Journal of Plant Biology
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    • v.30 no.2
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    • pp.109-116
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    • 1987
  • Effects of staining, buffer washing and denaturing agents on the transferrability of RNA fractionated on a methylmercury hydroxide-agarose gel to a nitrocellulose membrane were studied. Ethidium bromide staining and ammonium acetate buffer washing inhibited RNA transfer, while 3% HCHO and 0.5 M NaOH treatments stimulated transfer which was negated in the ammonium acetate buffer. Accordingly, maintenance of primary structure of RNA was proved to be essential for transferring RNA from the methylmercury hydroxideagarose gel to the nitrocellulose membrane.

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Anomalous Absorbance-Temperature Profile of Calf Thymus DNA in Presence of Spermine

  • Chan-Yong Lee;Hyeong-Won Ryu;Moon-Jip Kim;Thong-Sung Ko
    • Bulletin of the Korean Chemical Society
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    • v.12 no.3
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    • pp.262-264
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    • 1991
  • An anomalous absorbance-temperature profile of calf thymus DNA, having a hypochromic trough just before the rise the $T_m$-region phase, occurs at the spermine concentration where the DNA collapses into a compact structure. The trough phase can be eliminated by the addition of ethidium bromide and also by a hydrophobic environment.