• Journal of Microbiology
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• 제45권6호
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• 대한기계학회논문집A
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• 제32권10호
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• pp.844-849
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• 2008

We present a novel cell deformability monitoring chip based on the digitally measured cell lysis rate which is dependent on the areal strain of the cell membrane. This method offers simple cell deformability monitoring by automated high-throughput testing system. We suggest the filter design considering the areal strain imposed on the cell membrane passing through the filter array having gradually increased orifice length. In the experiment using erythrocytes, we characterized the cell deformability in terms of average fracture areal strain which was $0.24{\pm}0.014\;and\;0.21{\pm}0.002$ for normal and chemically treated erythrocytes, respectively. We also verified that the areal strain of 0.15 effectively discriminates the deformability difference of normal and chemically treated erythrocytes, which can be applied to the clinical situation. We compared the lysis rates and their difference for the samples from different donors and found that the present chips can be commonly used without any calibration process. The experimental results demonstrate the simple structure and high performance of the present cell deformability monitoring chips, applicable to simple and cost-effective cell aging process monitoring.

• Bulletin of the Korean Chemical Society
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• 제18권7호
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• pp.761-766
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• 1997

Phospholipase C from Clostridium perfringens is known to catalyze the hydrolysis of phospholipids in biological membranes. In this study, a simple and sensitive method for assaying phospholipase C was developed by using liposomes entrapping calcein as a fluorescent marker. Phospholipase C-induced lysis of liposomes was determined by measuring the fluorescence intensity of calcein released out from liposomes, Various liposomes with different compositions were prepared by reverse-phase evaporation method to investigate the effect of liposomal composition on the lytic activity of phospholipase C. The calcein-entrapping efficiency of liposomes was affected by the chain length of fatty acid in phosphatidylcholine constituting liposomes. The lytic activity of phospholipase C was the highest against liposomes prepared with eggPC. The lytic activity decreased with increasing chain length of fatty acid in phosphatidylcholine. Incorporation of cholesterol more than 20% into the liposomal bilayer inhibited the phospholipase C-induced lysis. The lysis of liposomes was more greatly increased by the addition of 10 mM of calcium. The lytic activity of phospholipase C was also affected by the surface charge of liposomes. Taken together, it was concluded that reverse-phase evaporation vesicles composed of dipalmitoylphosphatidylcholine and cholesterol in the molar ratio of 9 : 1 allowed to detect the lowest concentration of phospholipase C (0.10 μg/assay volume). This study suggested that the use of liposomes can provide a simple, sensitive and inexpensive method for assaying phospholipase C.

• BMB Reports
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• 제40권3호
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• pp.444-447
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• 2007

Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.

• Journal of Plant Biology
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• 제30권3호
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• pp.201-203
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• 1987

Total RNA was isolated from two months old wheat, rice, tobacco and sweet potato. The procedure used was simple and provided pure RNA preparation. Lysis of plant tissue in a buffer with guanidine thiocyanate and CsCl density gradient centrifugation separated RNA from the rest of the cellular components. Subsequent cholroform/1-butanol extraction and ethanol precipitation were necessary to ensure contaminant-free RNA preparation. Storage of the lysed plant tissue in the buffer with guanidine thiocyanate preserved the sample for two months without noticeable RNA degradation.

• 공업화학
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• 제9권6호
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• pp.857-863
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• 1998

Beer의 법칙을 이용하여 lysozyme-M. lysodeikticus cell의 용해반응속도 및 lysozyme의 활성을 측정할 수 있는 간단하고 새로운 실험방법을 제시하였다. M.lysodeikticus cell의 농도에 따라 용액의 UV 투과도를 측정하였으며 이것은 Beer의 법칙에 따르는 것으로 나타났다. 또한 용액 내의 lysozyme 농도 및 용해반응에 의해 생성되는 생성물은 UV 투과도에 전혀 영향을 미치지 않는 것으로 실험적으로 증명되었다. 따라서 lysozyme의 용해반응이 진행되는 동안 용액내에 존재하는 M. lysodeikticus cell의 농도는 UV-분광광도계를 이용하여 in-situ로 측정될 수 있었다. 이렇게 얻어진 M. lysodeikticus cell의 농도변화를 Michaelis-Menten식을 이용하여 lysozyme-M. lysodeikticus cell의 용해 반응속도 상수들을 결정하였다. 최대반응속도상수 $k_3$은 약 $0.1734sec^{-1}$로 나타났으며 Michaelis-Menten 상수는 약 $9.83{\times}10^{-6}M$으로 나타났다. Lysozyme의 용해반응속도가 활성에 의존하므로 이와 같은 측정을 통해 lysozyme의 활성도도 측정할 수 있었다.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.289-296
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• 1985

본 연구는 유산간균 및 유산구균으로부터 plasmid DNA를 신속하고 간편하게 분리하기 위한 방법에 관한 것이다. 세포벽 형성 억제 인자인 glycine을 0.5% 첨가한 TCM 배지에서 유산균을 배양하였고 plasmid DNA는 mutanolysin을 처리한 cells로부터 alkaline-detergent lysis 법으로 분리되었다. 유산간균은 효소 처리 때 mutanolysin의 농도를 30$\mu\textrm{g}$/$m\ell$로 하고 37$^{\circ}C$에서 5-10분간 반응되었을 때 plasmid DNA가 아주 잘 추출되었다. 유산구균의 경우는 그 최적 조건이 조금 달랐다. 본 방법은 L. casei, L. acidophilus, L. helveticus, S. lactis, S. faecalis, S. faecium과 S. cremoris 균주로부터 plasmid DNA를 신속하게 분리하는데 사용할 수 있었다. 본 방법을 이용하여 배양액 $1.5m\ell$로부터 분리된 plasmidsDNA가 gel상에서 쉽게 확인될 수 있었다.

• 대한기계학회:학술대회논문집
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• 대한기계학회 2008년도 추계학술대회A
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• pp.1752-1757
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• 2008

Simple and highly efficient droplet merging method is proposed, which enables two nanoliter or picoliter droplets to merge regularly in a straight microchannel. We observe that two droplets of the same size but of different viscosities are merged by velocity difference induced as they are transported with the carrier fluid. To make viscosity difference, the mass ratio of water and glycerol is varied. Two droplets of the same size or of different sizes are generated alternatingly in the cross channel by controlling flowrates. This droplet merging method can be used to mix or encapsulate one target sample with another material, so that it can be applied to cell lysis, particle synthesis, drug discovery, hydrogel-bead production, and so on.

• Tuberculosis and Respiratory Diseases
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• 제40권1호
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• pp.43-51
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• 1993

연구배경 : 결핵은 아직도 한국에서는 중요한 질병의 하나이지만 결핵을 진단하기 위한 보편적인 방법들이 단점인 예민도와 검사기간등이 문제가 되고 있다. 최근 분자유전학에 PCR 기법이 도입된 이래 결핵균의 DNA를 추출하여 짧은 시간내에 결핵을 진단하고자 하는 많은 보고들이 있어 왔다. 그러나 보고된 방법들의 다양성과 각 방법에 따른 소요시간등이 매우 다른 것이 문제가 되고 있고 임상에서 빠른 시간내에 비교적 간단한 방법으로 결핵율의 DNA를 분리한다면 보다 빨리 결핵을 진단하여 치료를 할 수 있다고 생각되어 결핵균의 DNA를 분리하는 방법중 가장 좋은 방법을 확인하고자 하였다. 방법 : 결핵율의 DNA를 추출하는 방법은 총 6가지의 방법으로서 SDS-microwave oven method, NaOH lysis method, Triton X-100-proteinase K method, Lysis buffer method, SDS-proteinase K method 및 Bead beater method를 사용하여 비교하였으며 사용된 균주는 Mycobacterium tuberculosis $H_{37}Rv$ strain 이었고 임상가검물중 도말양성 객담 5예 및 결핵성 뇌막염이 의심되고 도말음성이었으나 항결핵제 투여로 완치판정을 받은 환자의 뇌척수액 5예를 대상으로 하였다. 사용된 primer는 IS6110의 123bp를 target DNA를 하였고 PCR반응은 각각 $94^{\circ}C,\;68^{\circ}C$ 및 $72^{\circ}C$ 로 각각 2분씩 총 30주기를 시행하였다. 결과 : $H_{37}Rv$ strain에서 각 방법으로 추출된 DNA를 10배씩 serial dilution하여 PCR을 시행한 결과 SDS-microwave oven법과 NaOH lysis법은 50 fg까지 양성이었으며 나머지 4가지 방법에서는 5fg까지 양성이었다. 또한 각각의 방법으로 도말양성 환자의 객담에서 PCR을 시행한 결과 전예에서 양성이었고 결핵성 뇌막염이 의심스러운 환자의 뇌척수액에서는 SDS-microwave법과 NaOH lysis법으로는 5예중 3예에서 양성이었고 나머지 4가지 방법으로는 5예중 4예에서 양성이었다. 결론 : 이상의 실험으로 경제적이고 간편한 측면에서 볼 때 SDS-proteinase K법이 가장 좋은 방법임을 알 수 있었다.

• BMB Reports
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• 제37권3호
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• pp.351-355
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• 2004

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a singe tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.