• Title/Summary/Keyword: DNA extraction buffer

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Isolation of High-Quality mRNA from Tannin-Rich Persimmon Fruit (고 Tannin 함유 감과실로 부터 mRNA의 분리)

  • ;Dav
    • Food Science and Preservation
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    • v.4 no.1
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    • pp.45-51
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    • 1997
  • In our studies on the role of $\beta$-galactosidase in fruit softening, significant difficulty, was encountered in our attempts to extract RNA from persimmon(Diospyros kaki L. cv. Fuyu) fruit due to astringency and tannin content. Initial, unsuccessful RNA extractions involved methods using guanidinium isothiocyanate/CsCl with and without polyvinylpyrrolidone(PVP), phenol/sodium lauryl sulfate(SDS), guanidinium hydrochloride, as well as polysomal RNA purification method that used 0.2 M Tris-HCI (pH 9.0) containing KCI, Mg-acetate, EDTA, $\beta$-mercaptoethanol, and sucrose. A method was devised which employed treatment of fruit with CO2 gas to diminish astringency prior to RNA extraction, followed by extraction of tissue powders with Proteinase K extraction buffer containing PVP and ascorbate at an alkaline pH. This procedure resulted in the removal of tannins and other polyphenolics and extraction of relatively large amount of high-quality RNA suitable for cDNA library construction and polymerase chain reaction(PCR). Futhermore, the procedure does not use the toxic and corrosive chemical guanidinium isothiocyanate or require ultracentrifugation.

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Transformation Conditions of Bacillus subtilis by Streptomyces rimosus Plasmid DNA (Streptomyces rimosus Plasmid DNA에 의한 Bacillus subtilis의 형질전환 조건)

  • Hong, Yong-Ki;Seu, Jung-Hwn
    • Microbiology and Biotechnology Letters
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    • v.11 no.1
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    • pp.75-79
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    • 1983
  • To exploit a suitable vector and recipient strain for molecular cloning in Bacillus subtilis, oxytetracycline-resistant plasmic DNA has been prepared from Streptomyces rimosus by phenol-buffer extraction of lysozyme-lysed cells and introduced into B. subtilis KPM 60 [St $r^{R}$-mutant of RM 125 (leu A8, arg 15, hsm $M^{-10}$ , hsr $M^{-10}$ )] by transformation. Oxitetracycline-resistant plasmid was well transferred into B. subtilis KPM 60 with average frequency of 10$^{-4}$ per $\mu\textrm{g}$ of DNA. The highest frequency of plasmid transformation was obtained after 3 hours incubation of recipient cells in the growth medium and 30 to 60 minutes incubation in the competence medium, and then 20 minutes contact of DNA and host cells. Optimum pH for competence was 7.5, and optimum temperature for transformation was 2$0^{\circ}C$.>.

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Rapid Isolation of Genomic DNA from Normal and Apoptotic Cells Using Magnetic Silica Resins

  • Park, Jee-Sun;Park, Jung-Hyun;Na, Shin-Young;Choe, Soo-Young;Choi, Sang-Nam;You, Kwan-Hee
    • Journal of Microbiology and Biotechnology
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    • v.11 no.5
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    • pp.890-894
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    • 2001
  • The isolation of genomic DNA from mammalian cells is usually performed by cell lysis followed by protein digestion, extraction, and finally, ethanol precipitation of the chromosomal DNA. However, in the case of large sample numbers or when only small amounts of starting materials are available, such conventional methods are not efficient and are cumbersome to be applied. Some alternative methods have been described as well as having commercial DNA isolation kits to be available, nevertheless, there is room left for much improvement. In the present study, a novel method is introduced, where it simplifies conventional protocols by omitting some time-consuming steps such as protease incubation or DNA precipitation and its resuspension. Using paramagnetic silica resins, the genomic DNA was purified over a magnetic field, and the bound DNA was eluted with a low-salt buffer. The fidelity and effectiveness of this novel method was determined by using normal and apoptotic cells as a starting material and then compared to other protocols. The high speed and convenience along with its high efficiency in detecting apoptotic chromosomal DNA will prove this method to be an improved alternative in the isolation of genomic DNA from mammalian cells.

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RAPD Analysis for Genetic Diversity of Melon Species (참외와 멜론의 유전적 다양성에 대한 RAPD 분석)

  • Mo, Suk-Youn;Im, Sung-Hee;Go, Gwan-DaI;Ann, Chong-Mun;Kim, Doo Hwan
    • Horticultural Science & Technology
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    • v.16 no.1
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    • pp.21-24
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    • 1998
  • RAPD markers were analyzed in order to detect the genetic variation and diversity of the fifty-two melon lines. SDS extraction method produced more and purer DNA than CTAB method. RAPD reaction conditions were optimized as follows ; 10ng template DNA, 270nM primer, $200{\mu}M$ each of dATP, dCTP, dGTP and dTTP, $0.3{\mu}unit$ dynazyme and 10x buffer brought to $15{\mu}l$ final volume with distilled water. The adequate annealing temperature was $39^{\circ}C$ and forty cycles of amplification produced the best RAPD band patterns. Among a total of 123 bands from 12 random primers, 25 polymorphic bands(20%) were selected as reliable markers. The average number of polymorphic bands per primer was 2.1 among the 52 lines. Intragroup genetic relationship based on the marker difference was closer than intergroup genetic relationship. The 52 lines could be grouped into two major group (Korean landraces and melon lines) and then melon group subdivided into two subgroups (net melon lines and no-net melon). This result corresponded to morphological grouping. Eight RAPD markers separated the Korean landraces and melon groups and four RAPD markers separated net melon and no-net melon groups.

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A Simple, Rapid, and Automatic Centrifugal Microfluidic System for Influenza A H1N1 Viral RNA Purification

  • Park, Byung Hyun;Jung, Jae Hwan;Oh, Seung Jun;Seo, Tae Seok
    • Proceedings of the Korean Vacuum Society Conference
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    • 2013.08a
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    • pp.277.1-277.1
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    • 2013
  • Molecular diagnostics consists of three processes, which are a sample pretreatment, a nucleic acid amplification, and an amplicon detection. Among three components, sample pretreatment is an important process in that it can increase the limit of detection by purifying nucleic acid in biological sample from contaminants that may interfere with the downstream genetic analysis such as nucleic acid amplification and detection. To achieve point-of-care virus detection system, the sample pretreatment process needs to be simple, rapid, and automatic. However, the commercial RNA extraction kits such as Rneasy (Qiagen) or MagnaPure (Roche) kit are highly labor-intensive and time-consuming due to numerous manual steps, and so it is not adequate for the on-site sample preparation. Herein, we have developed a rotary microfluidic system to extract and purify the RNA without necessity of external mechanical syringe pumps to allow flow control using microfluidic technology. We designed three reservoirs for sample, washing buffer, and elution buffer which were connected with different dimensional microfluidic channels. By controlling RPM, we could dispense a RNA sample solution, a washing buffer, and an elution buffer successively, so that the RNA was captured in the sol-gel solid phase, purified, and eluted in the downstream. Such a novel rotary sample preparation system eliminates some complicated hardwares and human intervention providing the opportunity to construct a fully integrated genetic analysis microsystem.

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Diagnosis of Potato Leafroll Virus with Digoxigenin-labeled cRNA Probes (Digoxigenin으로 표지된 cRNA 프로브를 이용한 감자잎말림바이러스(PLRV)의 짐단)

  • 서효원;함영일;오승은;신관용;최장경
    • Korean Journal Plant Pathology
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    • v.14 no.6
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    • pp.636-641
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    • 1998
  • Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

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IDENTIFICATION OF PUTATIVE PATHOGENS IN ACUTE ENDODONTIC INFECTIONS BY PCR BASED ON 16S rDNA (중합효소연쇄반응법을 이용한 급성 치수 및 치근단 질환의 병원성 세균의 동정)

  • Kim, Ji-Hoon;Yoo, So-Young;Lim, Sun-A;Kook, Joong-Ki;Lim, Sang-Soo;Park, Seul-Hee;Hwang, Ho-Keel
    • Restorative Dentistry and Endodontics
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    • v.28 no.2
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    • pp.178-183
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    • 2003
  • The purpose of this study was to investigate the frequency of 7 putative pathogens in endodontic infections. The specimens were collected from infected pulpal tissue of patients who were referred for root canal treatment to the department of conservative dentistry, Chosun University Samples were collected aseptically using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ml of 1 X PBS. DNAs were extracted from the samples by direct DNA extraction method using lysis buffer (0.5% EDTA, 1% Triton X-100). Identification of 7 putative pathogens was performed by PCR based on 16S rDNA. The target species were as follows : Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and Treponema denticola. Our data revealed that the prevalence of P. endodontalis was found in 88.6% (39/54), P. ginivalis 52.3% (23/44), P. nigrescens 18.2% (8/44), P intermedia 15.9% (7/44) B. forsythus 18.2% (8/44), A. actinomycetemcomitans 3.3% (1/44), T. denticola 25% (l1/44) of the samples. The high prevalence of P. endodontalis and P. ginivalis suggests that they may play an important role in the etiology of endodontic infections.

Detection of Megalocytivirus in shellfish using PCR with various DNA extraction methods (다양한 PCR용 DNA 추출법에 의한 패류 내 Megalocytivirus의 검출)

  • Kim, Jin-Woo;Cho, Mi-Young;Jin, Ji-Woong;Kim, Ki-Hong;Jeong, Hyun-Do;Kim, Kwang-Il
    • Journal of fish pathology
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    • v.24 no.2
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    • pp.65-73
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    • 2011
  • In analysis of DNA viruses from the contaminated shellfish using PCR, preparation method of template DNA is an important factor to get enough copy number of viruses. In this study, we evaluated the efficiency of PCR template of Megalocytivirus (sT50mg-D) DNA obtained from 50 mg digestive gland homogenate of oyster using commercial method, and compared with that obtained from 5 g of the same tissues (T5g-D) after PEG precipitation procedures of virus. Both templates DNA suspended in the same volume of distilled water showed positive results by primary PCR with 35 cycles, and the presence of Megalocytivirus was confirmed in oysters collected from cultured farms in Korea. Moreover, PCR with sT50mg-D allowed us to discriminate the contaminated oyster individually, that can not be done in PCR with T5g-D prepared from the mixture of three different individual oyster to get 5 g digestive gland homogenate. In quantitative analysis with real time PCR, Megalocytivirus concentrations in 50 ${\mu}l$ templates prepared using 0.5~50 mg of one positive sample were appeared in the range 6.14E+00~1.2E+02/${\mu}l$. We were not able to get positive result using template DNA contained less than 6.14E+00 copies. Consequently, 2-step PCR performed with DNA extracts from oyster homogenate of small amount (sT50mg-D) i) was enough to detect the contaminated Megalocytivirus in shellfish, ii) allowed us to do the analysis for individual shellfish rather than mixture of several shellfish and iii) showed the presence of Megalocytivirus in oyster from Korea.

A Study on The Content of Liver Protein, Nucleic Acids, and Guanine Deaminase Activity of Mouse During Acute Starvation (급성(急性) 기아(饑餓)마우스의 간단백질(肝蛋白質), 핵산(核酸) 및 Guanine Deaminase 활성(活性)에 관(關)한 연구(硏究))

  • Park, Seung-Hee;Kim, Seung-Won
    • Journal of Nutrition and Health
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    • v.1 no.2
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    • pp.107-115
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    • 1968
  • Number of aspects, not only nutritional but social as well as political involved in human starvation pose nowadays global problems. In order to help establish the minimum nutritional requirements in the daily life of a man and to free people as well from either undernourishment, malnutrition or even starvation many workers have devoted themselves so far on the research programs to know what and how number of metabolic events take place in animals in vivo. It is the purpose of the present paper to examine in effect to what extent both of the protein and nucleic acids (DNA & RNA) together with an enzyme, guanine deaminase, which converts guanine into xanthine and in turn ends up to uric acid as an end product, undergo changes, quantitatively during acute starvation, using the mouse as an experimental animal. The mouse was strictly inhibited from taking foods except drinking water ad libitum and was sacriflced 24, 48, and 72 hours following starvation thus acutely induced. The animals consisted of two experimental groups, one control and another starvation groups, each being consisted of 6-24 mice of whose body weights ranged in the vicinity of 10 g. The animals were sacriflced by a blow on the head, followed by immediate excision of their livers into ice-cold distilled water, washing adherent blood and other contaminant tissues. The liver was minced foramin, by an all-glass homogenizer immersing it in an ice-bath, followed by subsequent fractionatin of the homogenate (10% W/V in 0.25M sucrose solution made up with 0.05M phosphate buffer of pH 7.4). For the liver protein and guanine deaminase assay, the 10% homogenate was centrifuged at 600 x g for 10 minutes to eliminate the nuclear fraction; and for the estimation of DNA and RNA, the homogenate was prepared by the addition of 10% trichloroacetic acid in order to free the homogenate from the acid-soluble fraction, the remaining residue being delipidate by the addition of alcohol and dried in vacuo for later KOH (IN) hydrolysis. The changes in body and liver wegihts during acute starvation were checked gravimetrically. Protein contents in the liver were monitored by the method of Lowry et al; and guanine deaminase activities were followed by the assay of liberated ammonia from the substrate utilizing the Caraway's colorimetry. The extraction of both DNA and RNA was performed by the Schmidt-Thannhauser's method, which was followed by Marmur's method of purification for DNA and by Chargaff's method of purification for RNA. The determinations of both DNA and RNA were carried out by the diphenylamine reaction for the former and by the orcinol reaction for the latter. The following resume was the results of the present work. 1. It was observed that the body as well as liver weights fall abruptly during starvation, and that the loss of body weight showed no statistical correlation with the decreases in the content of liver protein. 2. The content of liver protein and activity of liver guanine deaminase activity as well decline dramatically, and the specific activities of the enzyme (activity/protein), however, decreased gradually as starvation proceeded. 3. Both of the nucleic acids, DNA and RNA, showed decrements in the liver of mouse during acute starvation; the latter, however, being more striking in the decline as compared to the former. 4. The decreases in the liver protein content as resulted from the acute starvation had no statistically significant correlation with the decrements of DNA in the same tissue, but had regressed with a significant statistical correlation with the fall of RNA in the tissue. 5. The decrease in the activity of guanine deaminase in the liver of mouse during acute starvation was functionally more proportional to the decrease in RNA than DNA, and moreover correlated with the changes in the content of the liver protein. 6. The possible mechanisms involved during in this acute starvation as bring the decreases in the contents of DNA, protein, and guanine deaminase were discussed briefly.

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