Water DNA Isolation Protocol--E.Z.N.A

合集下载

DNA的粗提取与鉴定

DNA Master™ Genomic DNA Purification Kit 500 isolations ------------------- F1005 Each system contains sufficient reagents for 500 isolations of genomic DNA .
DNA Purification Buffer DNA 纯化剂 RNase A (4mg/ml) RNA 消化酶 Protocol 技术指南
规格 50 ml 25 ml 10 ml 250μl －
数量 1 1 1 1 1
产品编号 P0201 P0301 P0401 A0501 —
◎ 可供使用 500 次的 DNA Master™ 基因组 DNA 提纯试剂盒------------------ F1005 每一试剂盒含足量试剂，可进行 500 次基因组 DNA 小规模分离实验.
从食品、饲料产品中提取 DNA，首先需要将样品粉碎或者研磨，用核酸裂解剂裂解细胞及其核酸；RNA 消化为可选步骤，如需要可在此时进行；细胞内的蛋白质可以通过沉淀除去。其次，沉淀 DNA 并除盐。最后，加 DNA 纯化剂并保温以纯化 DNA。使用本试剂盒提取的 DNA 可用于进一步的分子生物学操作，包括 PCR 扩增，限制性内切酶切割，以及核酸杂交等。
The kit for plant DNA purification contains following Buffers: Small-Scale Isolation (miniprep)
DNA Master™ Genomic DNA Purification Kit 100 isolations ------------------ F1001 Each system contains sufficient reagents for 100 isolations of genomic DNA. Includes:

红狮自动化系列操作员界面面板CR1000和CR3000说明书

CR1000 and CR3000 HMIsRed Lion Automation SeriesPRODUCT HIGHLIGHTS>NEMA 4X, IP66 Front Panel Rating >Industrial Environmental Specifications •Wide operating temperature•High shock and vibration tolerance •CE, UL/cUL, UL/cUL Hazardous >Supports over 300 Industrial Drivers >SD Card for database upload/download >Built-In Web Server for Remote Access* >Real-Time Data Logging*CR1000 and CR3000 HMIs provide industry-leading protocol conversion and connectivity choices along w ith enhanced visual display features to deliver operator interface devices that easily scale and adapt as requirements change.The CR1000 and CR3000 automation HMIs from Red Lion combine an ever-expanding list of over 300 industrial drivers with the intuitive and powerful Crimson 3.1 development platform to create an operator experience designed to easily adapt as needs change and operations grow. Basic requirements for machine interface and protocol conversion are easily handled by the CR1000 products, while the CR3000 HMIs address more advanced needs with real-time data logging and a built-in web server.INDUSTRY APPLICATIONS >Factory Automation >OEM Machine Builders >Automotive >Food & Beverage >Water/WastewaterCUSU LFEATURES & BENEFITS > F ull-color HMI touchscreen•A variety of 4.3” to 15” models with widescreen(model dependent) ensures the right fit for virtually any application •16M colors>Industry-leading protocol conversion • C ommunicate with over 300 protocols• E asily convert between serial, USB and Ethernet devices•Convert up to 6 protocols simultaneously with CR1000 and up to 15 with CR3000•Manage multi-vendor environments with ease>Powerful Integration Capabilities• C rimson 3.1 software enables point-and-click configuration• U pdated 3D symbols and primitives create a modern operator experience •Ethernet, USB and serial ports make communication simple• R obust web server* provides remote access and control to reduce costly site visits•Send email and/or SMS notifications on alarm conditions*•Built-in data logging enhances troubleshooting and helps meet regulatory requirements•Automate population of data values using SQL Queries feature**Model dependentCOMMUNICATION PROPERTIESUSB PortsUSB Device:CR1000 and CR3000 (All Models) - (1) USB DeviceT ype B adheres to USB specification 1.1USB Host:CR3000 (4.3”) – (1) USB Host portCR3000 (7”, 10.4”, 15”) – (2) USB Host portsType A adheres to USB specification 2.0 and supports full-speeddata transfers with hardware overcurrent protected (0.5 A max perport)Serial Ports for programming or communications: Format/Baud ratesindependently configurableCR1000 (All) - One (1) RS232 and One (1) RS232/RS422/RS485selectableCR3000 (4.3”) - Two (2) RS232 and One (1) RS422/RS485CR3000 (7”, 10.4”, 15”) - Two (2) RS232 and Two (2) RS422/RS485 Ethernet Ports: 1500 Vrms network isolationCR1000 (All) - One (1) 10/100 BASE-T(X) port (RJ45)CR3000 (4.3”) - One (1) 10/100 BASE-T(X) port (RJ45)CR3000 (7”, 10.4”, 15”) - Two (2) 10/100 BASE-T(X) ports (RJ45) POWER INPUTCR1000 (All models) 24 VDC +/- 20%CR3000 (All models) 10 to 30 VDCMust use a Class 2 circuit according to National Electrical Code (NEC),NFPA-70 or Canadian Electrical Code (CEC), Part I, C22.1 or a Limited Power Supply (LPS) according to IEC 60950-1 or Limited-energy circuit according to IEC 61010-1. Power connection via removable threeposition terminal block.Battery: Lithium coin cellTypical lifetime of 5 yearsPOWER CONNECTIONHigh compression cage-clamp terminal blockWire Strip Length: 0.3” (7.5 mm)Wire Gauge Capacity: 12 to 24 AWG (3.31 to 0.20 mm2) copper wire Torque: 4.4-5.3 inch-lbs (0.5-0.6 N-m)NETWORK MEDIA10BaseT: ≥ Cat3 cable100BaseTX: ≥ Cat5 cableENVIRONMENTALOperating Temperature: -10°C to 50°CStorage Temperature: -20°C to 70°COperating Humidity: 0 to 85% (non condensing)Operating Altitude: Up to 2000 metersShock: 30 g per IEC 68-2-27Vibration: 2 g @ 5-500 Hz per IEC 68-2-6CERTIFICATION & COMPLIANCECE ApprovedImmunity: IEC/EN 61000-6-2 for Industrial LocationsEmissions: IEC/EN 6100-6-4 for Industrial Locations; CISPR 11 Class A Safety: IEC/EN 61010-1RoHS CompliantUL ListedClass I, Division 2, Groups A, B, C and D for CR3000 onlyType 4X Indoor / IP66 Enclosure rating (front face only) MECHANICALConstruction: Polycarbonate enclosure with NEMA 4X/IP66 rating forindoor use when correctly fitted with the gasket provided. Installation Category II, Pollution Degree 2.Weight:CR1000 4.3” model – 15.0 oz (425 g)CR1000 7” model – 1.91 lb (868 g)CR1000 10.4” model – 3.08 lb (1.395 Kg)CR3000 4.3” model – 1.00 lb (454 g)CR3000 7” model – 2.01 lb (913 g)CR3000 10.4” model – 3.16 lb (1.435 Kg)CR3000 15” model – 4.12 lb (2.155 Kg)Specifications are subject to change. Visit for more information.Dashes in listed model numbers added to improve readability, and are not included in orderable part numbers. CR1000 and CR3000 HMIs Ordering Guide4.3” models7” Models10” ModelsTHIS VIEW SHOWN WITH MOUNTING PLATE AND BRACKETSTHIS VIEW SHOWN WITH MOUNTING PLATE AND BRACKETSConnect. Monitor. Control.ADLD0478 042718 © 2018 Red Lion Controls, Inc. All rights reserved. Red Lion, the Red Lion logo, N-Tron and Sixnet are registered trademarks of Red Lion Controls, Inc. All other company and product names are trademarks of their respective owners.As the global experts in communication, monitoring and control for industrial automation and networking, Red Lion has been delivering innovative solutions for over forty years. Our automation, Ethernet and cellular M2M technology enables companies worldwide to gain real-time data visibility that drives productivity. Product brands include Red Lion, N-Tron and Sixnet. With headquarters in York, Pennsylvania, the company has offices across the Americas, Asia-Pacific and Europe. Red Lion is part of Spectris plc, the productivity-enhancing instrumentation and controls company. For more information, please visit .THIS VIEW SHOWN WITH MOUNTING PLATE AND BRACKETSTHIS VIEW SHOWN WITH MOUNTING PLATE AND BRACKETS4.3” models7” Models10” Models。

endofree-质粒提取

Store diluted DNA Wash Buffer at room temperature Note: All Centrifugation steps must be carried out at room temperature.
Introduction
The E.Z.N.A.TM Family of products is an innovative system that radically simplifies extraction and purification of nucleic acids from a variety of sources. Key to the system is the Omega Biotek’s proprietary HiBindTM matrix that avidly, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or low salt buffer. Application such as transfection normally requires large quantity of plasmid that need a number of stringent criteria, including high degree of purity and high percentage of supercoiling. The most common difficulty for large scale plasmid isolation is the contaminant of endotoxin which can have a negative effect on downstream applications such as transfection. The E.Z.N.A.® Endo-free Plasmid Mini Kit combines the power of HiBind technology with the time-tested consistency of alkaline-SDS lysis of HiBind Maxi-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. Following the lysis, the cleared lysate is extracted with ETR Solution which will almost completely remove the endotoxin. Then the lysate is applied to the HiBind Mini-column, plasmid DNA is bound to the silica membrane and contaminants are removed with a simple wash step.Yield vary according to plasmid copy number, E.coli strain and conditions of growth, but up to 30 ìg of high quality plasmid can be purified from 5ml overnight culture. The product is suitable for automated fluorescent DNA sequencing, restriction endonuclease digestion, transfection of mammalian cells, and other manipulations.

wizard plus midipreps dna purification system protocol

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TB173Revised 12/10Page 11.Description (1)2.Product Components and Storage Conditions (2)3.Production of a Cleared Lysate (2)4.Plasmid DNA Purification (4)5.Supplementary Information (6)A.Factors Affecting Plasmid DNA Yield (6)B.Choosing a Bacterial Strain (6)C.Special Considerations for Automated Fluorescent Sequencing (7)6.Troubleshooting (9)position of Buffers and Solutions (11)8.Related Products (12)9.References (12)1.DescriptionSmall-scale purifications of plasmid DNA, known as minipreps, are commonly used in molecular biology procedures. Over the years many miniprep protocols have been used, but few have proven to be consistently reliable (1). The strategy has been improved and adapted for processing larger culture volumes (10–100ml) with the Wizard ®Plus Midipreps DNA Purification System (a).The Wizard ®Plus Midipreps DNA Purification System eliminates many of the problems associated with standard midiprep procedures and provides a simple and reliable method for rapid isolation of plasmid DNA. This system can be used to isolate any plasmid but works most efficiently with plasmids <20,000bp.When using the standard protocol, the entire midiprep process can be completed in 90 minutes or less with no organic extractions or ethanol precipitations. Multiple midipreps can be easily processed at one time with the Vac-Man ®(20-sample capacity, Cat.# A7231) or Vac-Man ®Jr. (2-sample capacity, Cat.# A7660) Laboratory Vacuum Manifold. DNA is eluted from the Wizard Midicolumn in Nuclease-Free Water (Cat.# P1193). The purified plasmid can be used directly for automated fluorescent DNA sequencing or Wizard ®Plus Midipreps DNA Purification SystemAll technical literature is available on the Internet at /tbs Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useof this system. E-mail techserv@.restriction digestion without further manipulation and also can be used forin vitro transcription reactions supplemented with a ribonuclease inhibitor, such as Recombinant RNasin®Ribonuclease Inhibitor (Cat.# N2511).2.Product Components and Storage ConditionsProduct Size Cat. # Wizard®Plus Midipreps DNA Purification System25 preps A7640 Each system contains sufficient reagents and columns for 25 isolations from 10–100ml of bacterial culture (using EndA– strains). Includes:•75ml Cell Resuspension Solution (CRA)•75ml Cell Lysis Solution (CLA)•150ml Neutralization Solution (NSA)•250ml Wizard®Midipreps DNA Purification Resin•355ml Column Wash Solution* (CWB)•25Midicolumns with Reservoirs*Column Wash Solution is provided in two bottles, one containing 125ml and the other 230ml of Column Wash Solution.Product Size Cat.# Wizard®Midipreps DNA Purification Resin*(a)1,000ml A7701 Wizard®Midicolumns100 each A7651Storage Conditions and Stability:Store the Wizard®Plus Midipreps DNAPurification System at room temperature (15–30°C). No refrigeration is required.Protect the resin from exposure to direct sunlight.3.Production of a Cleared LysateStart each Wizard®Plus Midiprep with a 10–100ml overnight culture of E. coli.DNA yields may vary between 10µg and 200µg depending on the volume ofbacterial culture, the plasmid copy number and the bacterial strain used. Up to200µg of high-copy-number plasmid DNA can be obtained from a 100mlculture. When isolating DNA from low-copy-number plasmids, it isrecommended to process 100ml of bacterial culture.Materials to Be Supplied by the User(Solution compositions are provided in Section 7.)•centrifuge capable of 10,000–14,000 × g•Miracloth™ (Calbiochem Corp. Cat.# 475855, filter paper (Whatman®#1, GFA or GFC) or an autoclaved coffee filter•ethanol (95%)Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TB173Printed in USA. Page 2Revised 12/10Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TB173Revised 12/10Page 3Before you begin , dilute both bottles of Column Wash Solution (CWB) asfollows:Add 320ml of 95% ethanol to the large bottle (230ml) for a final volume of550ml; add 170ml of 95% ethanol to the small bottle (125ml) for a final volume of 295ml.Note: Throughout the remainder of this document Column Wash Solution(CWB), Cell Resuspension Solution (CRA), Cell Lysis Solution (CLA) andNeutralization Solution (NSA) are referred to simply as Column Wash Solution,Cell Resuspension Solution, Cell Lysis Solution and Neutralization Solution.1.Pellet 10–100ml of cells by centrifugation at 10,000 × g for 10 minutes at4°C. Pour off the supernatant and blot the tube upside down on a papertowel to remove excess liquid.pletely resuspend the cell pellet in 3ml of Cell Resuspension Solution.(To aid resuspension, manually disrupt the pellet with a 12-inch applicatorstick or by pipetting until no clumps are visible. Complete resuspension iscritical for optimal yields.)3.Add 3ml of Cell Lysis Solution and mix by inverting the tube four times.Do not vortex. The cell suspension should clear immediately.Note:Some bacterial cells are more resistant to lysis and may requireincubation for 3–5 minutes for efficient lysis. Additionally, culture volumes>50ml will take an extra 3–5 minutes to clear. The lysate may not appearcompletely clear, but do not extend lysis time beyond 3–5 minutes as thismay result in the formation of single-stranded DNA in the preparation.4.Add 3ml of Neutralization Solution and mix by inverting the tube 4 times.Alternatively , if using an EndA+ strain, add 6ml of NeutralizationSolution, mix by inverting the tube 4 times, and incubate the lysate at roomtemperature for 10 minutes. Proceed to Step 5.5.Centrifuge at 14,000 × g for 15 minutes at 4°C. If a tight pellet has notformed by the end of the centrifugation, centrifuge for another 15 minutes.6.Carefully decant the supernatant to a new centrifuge tube, avoiding thewhite precipitate. Alternatively, transfer the cleared supernatant by filteringit through Miracloth™ (Calbiochem Corp. Cat.# 475855), filter paper(Whatman ®#1, GFA or GFC) or an autoclaved coffee filter into the newcentrifuge tube. Proceed immediately to Section 4.4.Plasmid DNA PurificationMultiple Wizard ®Plus Midipreps can be easily processed simultaneouslywith the Vac-Man ®or Vac-Man ®Jr. Laboratory Vacuum Manifold, which is required for this procedure.Materials to Be Supplied by the User(Solution compositions are provided in Section 7.)•vacuum pump or vacuum aspirator capable of a vacuum of 15–18 inches of mercury (Hg)•vacuum manifold (i.e., Vac-Man ®(Cat.# A7231) or Vac-Man ®Jr. (Cat.# A7660) Laboratory Vacuum Manifold)•Nuclease-Free Water (Cat.# P1193) preheated to 65–70°C •optional:40% isopropanol/4.2M guanidine hydrochloridesolution (required for EndA+ strains—use only PromegaCat.# H5381)Thoroughly mix the Wizard ®Midipreps DNAPurification Resin before removing an aliquot.1.Add 10ml of resuspended Wizard ®Midipreps DNA Purification Resinto the DNA solution from Section 3. Swirl to mix.Note:Extended incubation of the resin and lysate is not necessary. Donot allow the resin to remain in contact with the lysate for longer than ittakes to load the Midicolumns.2.For each Midiprep, use one Midicolumn. Insert the Midicolumn tip intothe vacuum manifold port.3.Transfer the resin/DNA mixture into the Midicolumn. Apply a vacuumof at least 15 inches of Hg to pull the resin/DNA mix into theMidicolumn. When all of the sample has passed through the column,break the vacuum at the source.If using an EndA+ strain:a.The total volume of lysate and resin will exceed the column capacity by1ml. Therefore, all but 2–4ml of the resin/DNA should be applied to thecolumn. After the vacuum is applied and the column volume drops, theremainder of the lysate and resin can be added.b.Add 15ml of 40% isopropanol/4.2M guanidine hydrochloride solution(Section 7) to each column. Apply a vacuum continuously until 30seconds after all of the solution has flowed through the columns. Notethat this solution will flow through the column more slowly than thestandard Column Wash Solution. Proceed to Step 4.4.Add 15ml of Column Wash Solution to the Midicolumn and apply avacuum to draw the solution through the Midicolumn.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TB173Printed in USA.Page 4Revised 12/105.Break the vacuum at the source and add another 15ml of Column WashSolution to the Midicolumn. Reapply a vacuum to draw the solutionthrough the Midicolumn.Note:The Column Wash procedure may take up to 30 minutes.6.Dry the resin by continuing to draw a vacuum for 30 seconds after thesolution has been pulled through the column. Do not dry the resin formore than 30 seconds.Remove the Midicolumn from the vacuum source.Separate the Reservoir from the Midicolumn by breaking or cutting withsharp scissors as shown in Figure 1. Transfer the Midicolumn to a 1.5mlmicrocentrifuge tube. Centrifuge the Midicolumn at 10,000 × g in amicrocentrifuge for 2 minutes to remove any residual Column WashSolution. Transfer the Midicolumn to a new microcentrifuge tube.7.Add 300µl of preheated (65–70°C) Nuclease-Free Water to the Midicolumnand wait 1 minute. Elute the DNA by centrifuging the Midicolumn at10,000 × g8.9.eluted DNA.Figure 1. Location of the cut site for separating the Reservoir from the Midicolumn.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TB173Revised 12/10Page 5Cut column here0991M A 08_3A5.Supplementary InformationPlasmid DNA can be purified from 10–100ml overnight cultures of E. coliwith the Wizard®Plus Midipreps System. The yield of plasmid will varydepending on a number of factors, including the volume of bacterial culture,plasmid copy number, type of culture medium and the bacterial strain. Theprotocol presented in this technical bulletin is for the isolation of plasmidDNA from E. coli.5.A.Factors Affecting Plasmid DNA YieldPlasmid copy number is one of the most important factors affecting yield ina given system. Copy number is determined primarily by the region ofDNA surrounding and including the origin of replication in the plasmid.This area, known as the replicon, controls replication of plasmid DNA bybacterial enzyme complexes. Some DNA sequences, when inserted into aparticular vector, can lower the copy number of the plasmid. In addition,excessively large DNA inserts can also reduce plasmid copy number.5.B.Choosing a Bacterial StrainEndonuclease I is a 12kDa periplasmic protein that degrades double-stranded DNA. This protein is encoded by the gene end A. The E. coligenotype end A1 refers to a mutation in the wildtype end A gene, whichproduces an inactive form of the nuclease. E. coli strains with this mutationin the end A gene are referred to as EndA negative (EndA–). Table 1 containsa list of EndA– and EndA+ E. coli strains. The wildtype is indicated asEndA+. Using the Wizard®Plus Midipreps System, high-quality DNA iseasily obtained from both EndA+ and EndA– strains. Special precautionsmust be taken when working with EndA+ strains to ensure the isolation ofhigh-quality DNA (2), including the use of several modified protocol steps,as indicated, and the use of a less rich growth medium (e.g., LB). Themodified protocol will eliminate most problems associated with thesestrains. However, the level of endonuclease I produced is strain-dependent,and the modified protocol may not totally exclude endonuclease I fromplasmid DNA prepared from very high endonuclease I-producing strains.Also note that the modified protocol requires the use of increased volumesof several of the supplied solutions and, as a result, you will be unable toperform as many isolations. In general, we recommend using EndA– strainswhenever possible.Figure 2 depicts DNA isolated from varying amounts of culture, using high-and low-copy-number plasmids and an EndA– E. coli strain with theWizard®Plus Midipreps DNA Purification System.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TB173Printed in USA. Page 6Revised 12/10Figure 2. Plasmid DNA yield as a function of culture volume and plasmid copynumber, determined by use of the Wizard ®Plus Midipreps DNA PurificationSystem.Representative yields of a high-copy-number plasmid, pGEM ®-3Zf(+)Vector, and a low-copy-number plasmid, pBR322, prepared in E. coli strain DH5α™.Cultures were grown overnight at 37°C in 25ml increments of LB mediumcontaining 100µg/ml ampicillin.Table 1. List of EndA– and EndA+ Strains.5.C.Special Considerations for Automated Fluorescent SequencingFor the application of automated fluorescent sequencing, special considerationshould be given to the selection of plasmid type and E. coli strain to optimizeyield and plasmid quality. Optimal automated fluorescent sequencing resultsare routinely obtained by using high-copy-number plasmids and EndA–strains of E. coli .Note:For fluorescent sequencing applications, elute and store the DNA innuclease-free water.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TB173Revised 12/10Page 725020015010050Culture Volume (ml)P l a s m i d Y i e l d (μg )1460M A 04_6APurified plasmid DNA must be within the proper concentration range forsuccessful automated cycle sequencing (ideally 0.2µg/µl, not less than0.1µg/µl).When working with plasmid DNA from low-copy-numberplasmids, we strongly recommend that DNA concentrations be determinedby agarose gel/ethidium bromide quantitation prior to any application.DNA quantitation by spectrophotometric methods is prone to errors andrequires a large amount of sample.The Wizard®Plus Midipreps System routinely yields 70µg of medium- orhigh-copy-number plasmid DNA when used with the pGEM®Vector andDH5α™ cells in 25ml of culture. For low-copy-number plasmids, a largerculture volume is required to obtain sufficient DNA for sequencing. Insome cases it may be possible to amplify plasmid DNA by growing thebacteria in the presence of antibiotics such as chloramphenicol orspectinomycin (1).Special Considerations for Sequencing Using BigDye®ChemistryWhen performing dilutions of BigDye™ terminator ready reaction mix, it isessential to use an appropriate dilution buffer, such as 250mM Tris-HCl (pH9.0), 10mM MgCl2.Table 2 outlines the amount of terminator-ready reaction mix and dilutionbuffer required to obtain the appropriate dilution for BigDye®terminatorreactions. For details on running these reactions, please refer to the protocolsupplied with the BigDye®terminator system. For each reaction, add thereagents in Table 2 to a separate tube.Table 2. Appropriate dilutions for BigDye™ Terminator Reactions.AmountComponent No Dilution1:21:41:6terminator-readyreaction mix*8.0µl 4.0µl 2.0µl1.3µldouble-strandedplasmid DNA template200–500ng200–500ng200–500ng200–500ngprimer 3.2pmol 3.2pmol 3.2pmol3.2pmoldilution buffer**0µl 2.0µl 3.0µl3.4µlNuclease-Free Waterto a final volume of20µl20µl20µl20µl**Dilution buffer is a 5X solution.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TB173Printed in USA. Page 8Revised 12/106.TroubleshootingFor questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at: . E-mail: techserv@Symptoms Causes and CommentsPoor cell lysis Too many bacterial cells in culture medium.Use LB medium to grow bacteria. The use ofrich media or excessive culture volumes maylead to a biomass value too high for completelysis. All media should contain antibiotics at theappropriate concentration.Poor resuspension of bacterial cell pellet. Thecell pellet must be thoroughly resuspendedprior to cell lysis. Pipet or disperse (using anapplicator stick) the pellet with CellResuspension Solution. No cell clumps shouldbe visible after resuspension.No plasmid DNA purified Ethanol not added to the Column WashSolution. Prepare the Column Wash Solution asinstructed before beginning the procedure.EndA+ strain of bacteria used. DNA appearsdegraded or lost upon incubation with Mg2+containing buffer, i.e., restriction enzyme buffer.Follow protocol modifications for EndA+strains of bacteria.Inaccurate quantitation of plasmid DNA yield.Quantitate plasmid DNA yield by agarose gel/ethidium bromide electrophoresis.DNA floats out of well Carryover of residual ethanol from Columnduring loading of agarose gel Wash Solution. Follow directions forappropriate drying of resin by vacuum andcentrifugation. If DNA has already been eluted,precipitate DNA and dry remaining ethanolfrom the DNA pellet prior to resuspension inNuclease-Free Water. Increase loading dyeconcentration by 2X.Low plasmid DNA yields Overgrowth of bacterial culture by nontrans-formed bacteria. Make certain that antibioticswere used in all media, both liquid and solid.Do not culture bacteria longer than 24 hours.Optimal culture length is 12–16 hours.Bacterial culture too old. Inoculate antibiotic-containing media with freshly isolated bacterialcolony from an overnight plate.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Printed in USA.Part# TB173 Revised 12/10Page 96.Troubleshooting (continued)Symptoms Causes and CommentsLow plasmid DNA yields (continued)Low-copy-number plasmid used. See Section5.A. Cultures should not exceed the maximumrecommended volumes per isolation.Precipitate formed in resin. Warm resin in 37°Cwater bath for 15–20 minutes. Gently swirl bottleto mix and allow to cool to 30°C prior to use.Resin fines in eluted DNA. Follow directions forremoval of resin fines from eluted DNA, i.e.,filtration and centrifugation. If DNA aggregatehas formed, heat in the presence of 1M NaCl toredissolve aggregate. Centrifuge to remove resinfines. Precipitate DNA with ethanol and washwith 70% ethanol to remove residual NaClbefore using in downstream applications.Overdrying of resin on vacuum source. Followdirections for drying on vacuum source. Do notdry for times longer than suggested.Wrong reagents used. Make certain that ColumnWash Solution is diluted with ethanol beforeuse. Note that Wizard®Plus and Wizard®PlusSV components are not interchangeable.Plasmid DNA yield not accurately quantitated.Use agarose gel/ethidium bromide quantitation.Nicking of plasmid DNA Overincubation during the alkaline lysis step.Total incubation of cell suspension with CellLysis Solution should not exceed 5 minutes.No results or poor results with Too little DNA was added to the sequencingautomated fluorescent sequencing reaction. Inoculate fresh LB medium with anewly isolated E. coli colony. Purify plasmidDNA and quantitate by agarose gel/ethidiumbromide electrophoresis.TE buffer was used for DNA elution. Ethanolprecipitate and resuspend pellet in Nuclease-FreeWater. (The EDTA in TE buffer can interferewith downstream applications by chelating Mg2+.)ABI PRISM®BigDye®chemistry was used. Use ofthis chemistry necessitates ethanol precipitationof eluted DNA prior to sequencing reaction.Plasmid concentration not accurately quantitated.Use agarose gel/ethidium bromide electro-phoresis to accurately quantitate plasmid DNA. Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 · Part# TB173Printed in USA. Page 10Revised 12/106.Troubleshooting (continued)SymptomsCauses and CommentsNo restriction digestionIncrease the amount of restriction enzyme used and/or the length of incubation time. Digest at the recommended temperature and in the optimal buffer for the restriction enzyme used.DNA degraded during restriction digestion due to use of EndA+ E. coli strain. Repurify DNA from fresh culture containing antibiotics. Follow instructions (Section 3 and 4) for EndA+ strains or use an EndA– strain of E. coli .Genomic DNA contaminationVortexing or overmixing after addition of the Cell Lysis Solution. Do not vortex samples after addition of Cell Lysis Solution to prevent shearing of genomic DNA.DNA yields on gel look low compared Traces of contaminants may be present in the to spectrophotometer readingseluted DNA, inflating the spectrophotometer readings. Phenol:chloroform extract andprecipitate DNA, then wash with 70% ethanol before repeating spectrophotometer readings. Alternatively, quantitate DNA by agarose gel/ ethidium bromide electrophoresis.7.Composition of Buffers and SolutionsPromega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·Printed in USA.Part# TB173Revised 12/10Page 11Cell Resuspension Solution (CRA)50mM Tris-HCl (pH 7.5)10mM EDTA 100µg/ml RNase A Cell Lysis Solution (CLA)0.2M NaOH 1%SDSNeutralization Solution (NSA)1.32M potassium acetate (pH 4.8)Column Wash Solution (CWB)80mM potassium acetate 8.3mM Tris-HCl (pH 7.5)40µM EDTA Add 320ml and 170ml of 95% ethanol to the large and small bottles,respectively, of Column Wash Solution (Section 3). Final ethanol concentration will be approximately 55%.(Component concentrations listed are for final solution with ethanol added.)TE buffer (1X)10mM Tris-HCl (pH 7.5)1mM EDTA 40% isopropanol/4.2M guanidine HCl66.9g guanidine hydrochloride (use only Promega Cat.# H5381)Prepare a 7M solution by dissolving the guanidine hydrochloride in 50–60ml of sterile, distilled water. This reaction is very endothermic; warming the mixture to 37°C (do not exceed 37°C) will speed the process. Bring to a final volume of 100ml with sterile, distilled water.Prepare the 40% isopropanol/4.2M guanidine HCl solution by combining 30ml of the 7M guanidine HCl solution with 20ml of isopropanol in a 50ml screw-cap tube and mixing thoroughly.Store at room temperature.(a)U.S. Pat. Nos. 5,658,548 and 5,808,041, Australian Pat. No. 689815 and European Pat. No. 0 723 549 have been issued toPromega Corporation for nucleic acid purification on silica gel and glass mixtures. Other patents are pending. © 2010 Promega Corporation. All Rights Reserved.pGEM, RNasin, Vac-Man and Wizard are registered trademarks of Promega Corporation. PureYield is a trademark of Promega Corporation.ABI PRISM and BigDye are registered trademarks of Applera Corporation. Luer-Lok is a registered trademark of Becton-Dickinson & Co. DH5αis a trademark of Life Technologies, Inc. Miracloth is a trademark of Calbiochem Corporation. Whatman is a registered trademark of Whatman Paper Company, Ltd.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.8.Related ProductsProductSize Cat.#PureYield™ Plasmid Midiprep System25 preps A2492100 preps A2495Wizard ®Plus SV Minipreps DNA Purification System 50 preps A1330(does not include Vacuum Adapters)250 preps A1460Cell Resuspension Solution 150ml A7112Cell Lysis Solution 150ml A7122Neutralization Solution 150ml A7131Column Wash Solution125mlA8102ProductSizeCat.#Vac-Man ®Laboratory Vacuum Manifold 20-sample capacity A7231Vac-Man ®Jr. Laboratory Vacuum Manifold 2-sample capacityA7660One-Way Luer-Lok ®Stopcocks10 eachA72619.References1.Ausubel, F.M. et al.(1989) Current Protocols in Molecular Biology , Vol. 2, John Wiley & Sons, New York.2.Schoenfeld, T. et al.(1995) DNA purification: Effects of bacterial strains carrying the end A1 genotype on DNA quality isolated with Wizard ®plasmid purification systems. Promega Notes 53, 12.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·Part# TB173Printed in USA.Page 12Revised 12/10。

如何对待疑似病人英语作文

如何对待疑似病人英语作文Handling Suspected Patients: A Comprehensive Approach。

In today's global landscape, especially with theongoing challenges posed by infectious diseases, dealingwith suspected cases requires a delicate balance of caution, empathy, and efficiency. As we navigate through uncertain times, it's crucial to adopt a systematic approach to manage suspected cases effectively. Here's how:1. Early Identification: Prompt identification of suspected cases is paramount. This involves recognizing symptoms commonly associated with the particular disease in question. Fever, cough, shortness of breath, fatigue, and body aches are typical indicators. Additionally, recent travel history or contact with confirmed cases should raise red flags.2. Isolation Protocol: Once a suspected case is identified, immediate isolation is necessary to preventpotential spread. Isolation facilities should be equipped with proper ventilation and protective gear for healthcare workers. The patient should be isolated in a designated area away from other patients and visitors.3. Communication and Education: Clear communication is essential to avoid panic and misinformation. Informing the suspected patient about the situation, necessary precautions, and the importance of isolation is crucial. Additionally, educating the public about symptoms, preventive measures, and when to seek medical help can help contain the spread.4. Testing and Diagnosis: Timely testing is vital to confirm or rule out the infection. Utilizing reliable diagnostic tests ensures accurate results. Rapid antigen tests or PCR tests are commonly used depending on availability and accuracy. However, while awaiting results, the suspected patient should continue to be isolated to prevent potential transmission.5. Contact Tracing: Identifying and monitoringindividuals who have been in close contact with the suspected case is imperative. Contact tracing helps breakthe chain of transmission by identifying potential secondary cases early on. These contacts should be advisedto self-isolate and monitor symptoms for a specified period.6. Medical Care and Support: Providing adequate medical care and support to suspected cases is essential for their well-being and recovery. Symptomatic treatment, such asfever management and respiratory support, should be administered as needed. Psychological support is alsocrucial to alleviate anxiety and stress associated with isolation and uncertainty.7. Follow-Up and Monitoring: Continuous monitoring of suspected cases is necessary to track their progress and detect any complications. Regular follow-up consultations with healthcare professionals ensure proper management and timely intervention if symptoms worsen.8. Discharge Protocol: Once the suspected case tests negative and symptoms resolve, a systematic dischargeprotocol should be followed. This involves ensuring the patient's fitness for discharge, providing guidance onpost-isolation precautions, and emphasizing the importanceof ongoing preventive measures.9. Data Sharing and Collaboration: Collaboration between healthcare facilities, public health authorities, and research institutions is vital for effective management of suspected cases. Sharing data on confirmed and suspected cases, as well as epidemiological information, facilitatesa coordinated response and informed decision-making.10. Preventive Measures: Finally, implementing preventive measures such as vaccination, hand hygiene,mask-wearing, and social distancing is crucial in reducing the risk of transmission and mitigating the impact of infectious diseases in the community.In conclusion, a comprehensive approach to handling suspected cases involves early identification, isolation, clear communication, testing, contact tracing, medical care, follow-up, discharge planning, collaboration, andpreventive measures. By adhering to these principles and protocols, we can effectively manage suspected cases and minimize the spread of infectious diseases within our communities.。

HPV文献

ORIGINAL CONTRIBUTIONPrevalence of HPV Infection Among Females in the United StatesEileen F.Dunne,MD,MPHElizabeth R.Unger,PhD,MDMaya Sternberg,PhDGeraldine McQuillan,PhDDavid C.Swan,PhDSonya S.Patel,BSLauri E.Markowitz,MDH U M A N P A P I L L O M A V I R U S(HPV)is estimated to be themost common sexuallytransmitted infection in theUnited States.1HPV prevalence has beenfound to be highest among young per-sons within the first few years aftersexual debut.2-6However,there are nodata on the prevalence of HPV amongwomen across a broad age range andrepresentative of the US population.Genital HPV types are categorized ac-cording to their epidemiological asso-ciation with cervical cancer.Infec-tions with low-risk types,such as HPVtypes6and11,can cause benign or low-grade changes in cells of the cervix,genital warts,and recurrent respira-tory papillomatosis.High-risk HPVtypes can cause cervical,anal,and othergenital cancers.High-risk HPV types aredetected in99%of cervical cancers,andworldwide approximately70%of cer-vical cancers are due to HPV types16 and18.7,8Although HPV infection is common,studies suggest approxi-mately90%of infections clear within 2years.9,10A highly efficacious prophylactic vaccine against HPV types6,11,16, and18was licensed in June2006and recommended for routine use in females aged11to12years in theUnited States.11-14Clinical studies ofthe quadrivalent HPV vaccine demon-strated close to100%efficacy in pre-venting infection and disease(cervicalcancer precursors,genital lesions)associated with types included in thevaccine in analyses restricted to thosewomen who were naive to HPV types6,11,16,or18(either by HPV DNAor HPV antibodies).Representative data on type-specific prevalence of HPV DNAdetection in the United States couldprovide a baseline estimate to mea-See also p876and Patient Page.Author Affiliations:Centers for Disease Control andPrevention,Atlanta,Ga(Drs Unger and Swan,and MsPatel);and National Center for Health Statistics,Cen-ters for Disease Control and Prevention,Bethesda,Md(Dr McQuillan).Corresponding Author:Eileen F.Dunne,MD,MPH,Centers for Disease Control and Prevention,1600CliftonRd,MS E-02,Atlanta,GA30333(dde9@). Context Human papillomavirus(HPV)infection is estimated to be the most com-mon sexually transmitted infection.Baseline population prevalence data for HPV in-fection in the United States before widespread availability of a prophylactic HPV vac-cine would be useful.Objective To determine the prevalence of HPV among females in the United States. Design,Setting,and Participants The National Health and Nutrition Examina-tion Survey(NHANES)uses a representative sample of the US noninstitutionalized ci-vilian population.Females aged14to59years who were interviewed at home for NHANES2003-2004were examined in a mobile examination center and provided a self-collected vaginal swab specimen.Swabs were analyzed for HPV DNA by L1con-sensus polymerase chain reaction followed by type-specific hybridization.Demo-graphic and sexual behavior information was obtained from all participants.Main Outcome Measures HPV prevalence by polymerase chain reaction. Results The overall HPV prevalence was26.8%(95%confidence interval[CI],23.3%-30.9%)among US females aged14to59years(n=1921).HPV prevalence was24.5% (95%CI,19.6%-30.5%)among females aged14to19years,44.8%(95%CI,36.3%-55.3%)among women aged20to24years,27.4%(95%CI,21.9%-34.2%)among women aged25to29years,27.5%(95%CI,20.8%-36.4%)among women aged 30to39years,25.2%(95%CI,19.7%-32.2%)among women aged40to49years, and19.6%(95%CI,14.3%-26.8%)among women aged50to59years.There was a statistically significant trend for increasing HPV prevalence with each year of age from14to24years(PϽ.001),followed by a gradual decline in prevalence through 59years(P=.06).HPV vaccine types6and11(low-risk types)and16and18(high-risk types)were detected in3.4%of female participants;HPV-6was detected in1.3% (95%CI,0.8%-2.3%),HPV-11in0.1%(95%CI,0.03%-0.3%),HPV-16in1.5% (95%CI,0.9%-2.6%),and HPV-18in0.8%(95%CI,0.4%-1.5%)of female par-ticipants.Independent risk factors for HPV detection were age,marital status,and in-creasing numbers of lifetime and recent sex partners.Conclusions HPV is common among females in the United States.Our data indi-cate that the burden of prevalent HPV infection among females was greater than pre-vious estimates and was highest among those aged20to24years.However,the preva-lence of HPV vaccine types was relatively low.JAMA.2007;297:©2007American Medical Association.All rights reserved.sure the wide-scale impact of the vac-cine for reducing infection and could help guide models evaluating impact and cost effectiveness.With wide-spread implementation of the pro-phylactic HPV vaccine,decreases in the prevalence of vaccine HPV types would be expected.To determine a prevaccine population-based preva-lence of cervicovaginal HPV in the United States,we performed HPV DNA testing on self-collected vaginal swabs among females participating in the National Health and Nutrition Examination Survey(NHANES) 2003-2004.METHODSStudy Populationand Study DesignN H A N E S i s c o n d u c t e d b y t h e National Center for Health Statistics, Centers for Disease Control and Pre-vention,and uses a representative sample of the US noninstitutionalized civilian population.The representative sample is obtained by using a com-plex,stratified,multistage probability sample design with unequal probabili-ties of selection to obtain a nationally representative sample.15Certain sub-groups of people,such as adolescents, non-Hispanic blacks,and Mexican Americans are oversampled.All females aged14to59years selected for NHANES2003-2004were eligible for participation in this study.Of2482 females aged14to59years who were interviewed at home for the2003-2004cycle,2387(96.2%)were exam-ined in a mobile examination center.A total of2026females(81.6%)submit-ted cervicovaginal swab specimens. Four hundred sixty-six females (23.0%)were considered nonre-sponders because they either submit-ted an inadequate swab specimen (n=105)or they did not submit a swab specimen(n=361).There were significant differences between nonre-sponders and responders on some demographic and behavioral variables (nonresponders were significantly more likely than responders to be of other race/ethnicity,to be younger [Ͻ40years],to be born outside theUnited States or Mexico,and to havenever had sex).Written informed con-sent was obtained from all partici-pants,and parental permission forthose females younger than18years.This human subjects research wasapproved by the Centers for DiseaseControl and Prevention institutionalreview board.Demographic and Behavioral DataRace and ethnicity were self-reportedinto categories,including non-Hispanic black,non-Hispanic white,and Mexican American.Poverty indexratio was calculated by dividing totalfamily income by the poverty thresh-old index,adjusted for family size atyear of interview.16Estimates of thetotal number of cases were generatedby multiplying the appropriate popu-lation size from the January2004monthly postcensal civilian noninsti-tutionalized population17by theweighted prevalence estimate amongfemales in the appropriate agecategory.Sex was defined as vaginal,oral,oranal sex.For those females who had atleast1lifetime sex partner,additionalquestions were asked on age of first sexand lifetime number of sex partners.Forthose females aged14to17years whowere sexually active,additional ques-tions about sexual behavior were asked,including age at first sex,number of life-time partners,and ever use of a con-dom.For women aged18years or olderwho were sexually active,additionalquestions were asked on number andgender of sex partners in the last12months and lifetime sex partners,andpast history of sexually transmitted in-fections.Specimen Collectionand ProcessingFemales aged14to59years wereasked to self-collect a cervicovaginalsample in the mobile examinationcenter.In brief,each female was givena collection device,which was a smallfoam swab on a plastic handle pack-aged in an individual reclosable plasticsleeve(Catch-All Sample CollectionSwabs Epicenter,Madison,Wis).Par-ticipants were instructed to wash theirhands before opening the swab,tohold the swab by the end of thehandle,to insert the foam swab intothe vagina similar to inserting a tam-pon,to gently turn the swab during acount of10,and to replace the swab inthe plastic sleeve,avoiding contactwith the external genitalia.Partici-pants took swabs and instructions intoa bathroom and collected the samplesin privacy.Swabs were given toNHANES personnel,stored at roomtemperature,and mailed to the Cen-ters for Disease Control and Preven-tion laboratory for processing.DNA IsolationDNA was extracted using slight modi-fications of the QIAmp Mini Kit pro-tocol(Qiagen,Valencia,Calif)within1month of sample collection.Briefly,swabs were incubated at56°C for atleast12hours in proteinase K lysis so-lution.One half of each sample wasadded to an equal volume of100%etha-nol and applied to each of2QIAmpMini columns for DNA isolation.Theeluates were collected,washed,andconcentrated in Microcon100concen-trators(Fisher Scientific,Pittsburgh,Pa),adjusting the final volume to100µL with deionized distilled water.Samples were tested immediately orstored at−20°C.For every40samples,a water blank was processed throughall steps of extraction to serve as a con-tamination control.HPV Genotyping TestHPV detection and typing was per-formed by using the Roche prototypeline blot assay(reagents provided as agift from Roche Molecular Systems Inc,Pleasanton,Calif).This assay uses HPVL1consensus polymerase chain reac-tion with biotinylated PGMY09/11primer sets and␤-globin as an inter-nal control for sample amplifica-tion.18,19FiveµL of the DNA was usedin the100-µL polymerase chain reac-tion.Amplicons(10µL)were evalu-ated for␤-globin and HPV bands withHPV PREVALENCE©2007American Medical Association.All rights reserved.1.5%agarose gel electrophoresis stained with ethidium bromide,and those am-plicons with an HPV band were hy-bridized to the typing strips.The first generation strip used from January2003 to April2004included probes for27 HPV types(6,11,16,18,26,31,33, 35,39,40,42,45,51,52,53,54,55, 56,57,58,59,66,68,73,82,83,and 84).The second generation strip used after April2004included additional types(61,62,64,67,69,70,71,72,81, 89,and IS39)but omitted HPV-57.20 Samples that did not hybridize the strip were sequenced as previously de-scribed to determine HPV type.21 Samples negative for both␤-globin and HPV(n=105,5.2%)were considered inadequate for interpretation and were omitted from further analysis.We con-sidered low-risk HPV types as HPV type 6,11,32,40,42,44,54,55,61,62,64, 71,72,74,81,83,84,87,89,and91; and high-risk HPV types as HPV type 16,18,26,31,33,35,39,45,51,52, 53,56,58,59,66,67,68,69,70,73, 82,85,and IS39.Statistical AnalysisFemales who submitted an adequate swab for HPV evaluation were in-cluded in the final analysis(n=1921). Statistical analyses were conducted using SAS version9.122and SAS call-able SUDAAN.23Variance estimates were calculated by using a Taylor se-ries linearization that incorporated the complex sample design of the sur-vey.24All estimates were weighted using the2003-2004medical examination weights provided by National Center for Health Statistics to account for the un-equal probabilities of selection and ad-justment for nonresponse.The weight-ing methodology has been described previously.25Because there were some missing laboratory specimens,we in-vestigated whether any additional non-response adjustments to the original NHANES weights were needed.We found that using weights with an ad-ditional nonresponse and poststratifi-cation adjustment always provided prevalence estimates within the95% confidence interval(CI)based on the original NHANES weight;therefore,noadditional adjustments to the NHANESweights were made.We considered a prevalence esti-mate unreliable if the relative SE wasmore than30%of the prevalence esti-mate;these estimates are not pre-sented.Confidence intervals were cal-culated by using a log transformationwith the SE of the log prevalence basedon the delta method and applyingSUDAAN estimated SEs.26Tests of as-sociation between HPV and the demo-graphic or behavioral characteristicswere based on the Wald␹2statistic.Tocompare the prevalence between HPVtypes,we applied a version of theMcNemar test for complex surveys.Noadjustments were made to the P val-ues for multiple comparisons.To explore the association with ageand overall HPV prevalence,age was cat-egorized into4-year intervals(14-19,20-24,25-29,etc)and plotted against thelog odds of HPV.The plot revealed a non-linear association between age and HPVprevalence.Prevalence increased up toage20to24years and then decreased.To test for the presence of an increasinglinear trend among those femalesyounger than24years and a decreasinglinear trend for those females older than24years,2separate logistic regressionswere used.Each logistic regressiontreated age as a continuous variable andwas restricted to the age range under in-vestigation.A trend was considered sta-tistically significant if the␤coefficientfor the independant variable was non-zero at PϽ.05,using the Satterthwaite ad-justed F test.Demographic and behavioral char-acteristics of participants were evalu-ated.Characteristics significant at thePϽ.20level based on a Wald␹2statis-tic were considered in a multivariatemodel.There are only15df availableto develop a multivariate model,dueto the complex survey design in the2-year cycle of NHANES.To limit thedf used when including age in themodel,we collapsed age into4catego-ries(18-19,20-24,25-29,and30-59years),because the prevalences for agecategories between ages30and59years did not differ significantly.Themultivariate model was limited tofemales aged18to59years because allquestions regarding sexual exposureswere asked of this group.We usedSUDAAN for logistic regression tomodel independent associationsbetween prevalence of any HPV anddemographic and behavioral variablesamong sexually active females aged18to59years.We eliminated variables ina backward fashion that did not meetthe criteria of PϽ.05by Satterthwaiteadjusted F test at each step.Any par-ticipants with missing data on vari-ables included in the multivariateanalysis were excluded.Once all vari-ables in the model were statisticallysignificant at the PϽ.05level,all pair-wise interactions were evaluated andretained only if the overall P value forthe interaction wasϽ.05.Goodness offit for the final step of the model wasassessed using the Hosmer-LemeshowSatterthwaite adjusted F test.RESULTSHPV DNA Prevalence Overalland by AgeFrom2003-2004,2026vaginal swabspecimens from female NHANES par-ticipants aged14to59years were col-lected.Of the1921adequate speci-mens,26.8%(95%CI,23.3%-30.9%)were positive for any HPV ing2000Census data,this corresponds to24.9million females in this age rangewith prevalent HPV infection.Prevalence of HPV infection washighest among females aged20to24years(44.8%;95%CI,36.3%-55.3%);overall prevalence among females aged14to24years was33.8%(95%CI,28.6%-40.0%)(T ABLE1).There was astatistically significant trend for in-creasing HPV prevalence with each yearof age from14to24years(PϽ.001),which was followed by a nonsignifi-cant gradual decline in HPV preva-lence through59years(P=.06).When the analysis was restricted tosexually active females,the preva-lence of HPV was still highest amongthose aged20to24years.Among sexu-HPV PREVALENCE©2007American Medical Association.All rights reserved.ally active females,HPV prevalence was 39.6%(95%CI,32.9%-47.8%)for 14to 19years,49.3%(95%CI,40.7%-59.6%)for 20to 24years,27.8%(95%CI,21.7%-35.7%)for 25to 29years,27.3%(95%CI,20.2%-36.8%)for 30to 39years,23.9%(95%CI,18.8%-30.5%)for 40to 49years,and 20.2%(95%CI,14.6%-27.8%)for 50to 59years.Prevalence of High-Risk,Low-Risk,and Specific TypesThe overall prevalence of high-and low-risk HPV types was 15.2%and 17.8%,respectively.Prevalences of both low-risk and high-risk HPV types were high-est in females aged 20to 24years (F IGURE 1).There was a statistically sig-nificant difference between low-and high-risk HPV types among females aged 14to 19years and 50to 59years.Prevalence of high-risk types de-creased after 20to 29years,and preva-lence of low-risk types plateaued after 30to 39years.The most common HPV types de-tected were HPV-62(3.3%;95%CI,2.2%-5.1%)and HPV-84(3.3%;95%CI,2.2%-5.1%),HPV-53(2.8%;95%CI,2.1%-3.7%),and HPV-89(2.4%;95%CI,1.4%-4.3%)and HPV-61(2.4%;95%CI,1.6%-3.8%)(F IGURE 2).HPV-16was detected in 1.5%(95%CI,0.9%-2.6%)of females aged 14to 59years.There was no statistically signifi-cant difference in the prevalence of HPV-16and the 13more commonly de-tected types,except for HPV-84and HPV-62.HPV-6was detected in 1.3%(95%CI,0.8%-2.3%),HPV-11in 0.1%(95%CI,0.03%-0.3%;relative SE Ն30%),and HPV-18in 0.8%(95%CI,0.4%-1.5%)of female participants.Most participants infected with HPV (60.1%)had only 1HPV type detected (95%CI,53.2%-67.9%);however,23.9%had 2types (95%CI,18.3%-31.3%)and 16%had 3or more types detected (95%CI,12.0%-21.2%).Over-all,HPV types 6,11,16,or 18were de-tected in 3.4%of the study partici-pants,corresponding with 3.1million females with prevalent infection with HPV types included in the quadriva-lent HPV vaccine.Few participants (0.10%)had both HPV types 16and 18and none had all 4HPV vaccine types.At least 1of these 4HPV types was de-tected in 6.2%(95%CI,3.8%-10.3%)of females aged 14to 19years.Factors Associated With HPV DNA DetectionThe variables associated with HPV DNA detection that were significant in the bi-variate analysis were age,race,pov-erty index,education,marital status,and sexual behavior (Table 1and T ABLE 2).HPV DNA was detected in 5.2%of females who reported never having had sex (Table 2).In an unweighted analy-sis,88%of these females were be-tween 14to 19years,and the remain-ing 12%were 40to 49years.Two of these participants were also positive forFigure 1.Prevalence of Low-Risk andHigh-Risk HPV Types Among Females Aged 14to 59Years,NHANES 2003-200430-3940-4950-59Age, yP r e v a l e n c e , %14-1920-2425-29HPV indicates human papillomavirus;NHANES,Na-tional Health and Nutrition Examination Survey.Error bars indicate 95%confidence intervals.Both low-risk and high-risk HPV types were detected in some fe-males.Low-risk HPV types are defined as HPV type 6,11,32,40,42,44,54,55,61,62,64,71,72,74,81,83,84,87,89,and 91;and high-risk HPV types as HPV type 16,18,26,31,33,35,39,45,51,52,53,56,58,59,66,67,68,69,70,73,82,85,and IS39.Table 1.Prevalence of Any HPV Infection by Demographic Characteristics DemographicsSample SizePrevalence,%(95%Confidence Interval)P Value *Overall (aged 14-59years)192126.8(23.3-30.9)Age,y14-1965224.5(19.6-30.5)20-2418944.8(36.3-55.3)25-2917427.4(21.9-34.2).00330-3932827.5(20.8-36.4)40-4932425.2(19.7-32.2)50-5925419.6(14.3-26.8)Race†Non-Hispanic white 83724.2(20.5-28.6)Non-Hispanic black 53339.2(31.0-49.4)Ͻ.001Mexican American 44224.3(19.3-30.6)Marital statusMarried67617.3(14.0-21.5)Widowed,divorced,separated 23141.2(32.3-52.4)Never married 88231.1(28.1-34.5)Ͻ.001Living with partner 13246.1(35.2-60.4)Education‡ϽHigh school38335.0(29.4-41.7)High school and GED 38029.7(23.4-37.6).006ϾHigh school 75424.7(20.9-29.1)Poverty index§Below poverty50337.5(29.9-47.1).005At or above poverty 132224.4(21.1-28.4)Country of birthUnited States 158026.8(22.8-31.6)Mexico 20619.6(11.4-33.6).30Other ࿣13530.8(18.9-50.3)Abbreviations:GED,general equivalency diploma;HPV,human papillomavirus.*By Wald ␹2statistic.†Race and ethnicity were self-reported;other race (n =109)is not shown.‡This analysis was limited to females older than 17years.§Does not sum to 1921because some of the responses are missing.࿣Other includes any country other than the United States or Mexico.HPV PREVALENCE©2007American Medical Association.All rights reserved.©2007American Medical Association.All rights reserved.We found the overall prevalence of HPV-16to be low and that other HPV types were more prevalent.In most other studies,HPV-16has been found to be the most prevalent type,al-though prevalence varied based on the population evaluated.Population-based studies outside the United States have found lower prevalence of HPV-16 than clinic-based studies have.28It is possible that our cervicovaginal assess-ment using the self-collected vaginal swabs was more likely to detect HPV types not related to cervical infection. Castle et al28hypothesized that there is tropism of some phylogenetic group-ings(A3/A4/A15)to the vagina.In our assessment,the most common types de-tected were HPV-84and HPV-62,both in the A3phylogenetic grouping.Man-hart et al6found HPV types84and62 were also frequently detected in urine samples among females aged18to25 years.Independent risk factors for HPV DNA detection in our analysis in-cluded sexual behavior(number of sex partners in the last year,number of life-time sex partners)and demographicvariables,including young age andmarital status,consistent with risk fac-tors for HPV detection found in otherstudies.HPV DNA was detected in approxi-mately5%of women in our study whoreported never having had sex.In anunweighted analysis of these women,we found that most were young in ageand some had other sexually transmit-ted infections,suggesting their self-reported sexual history may not be ac-curate.Genital HPV is primarilyassociated with sexual intercourse;however,1study3found that nonpen-etrative sexual contact,such as genital-genital contact,could also result in HPVtransmission.A detailed sexual his-tory was not collected,therefore,wecould not evaluate specific types ofsexual contact.The84.9%response rate(number ofcollected swabs/number of eligibleswabs=2026/2387)for vaginal swabcollection in our study suggests that theself-collected swabs were a feasible andeffective method for HPV DNA detec-tion in a large survey.Previous assess-ments of the acceptability of self-collected vaginal swabs found thiscollection method to be more accept-able than specimens collected by healthcare professionals.29,30Evaluations havealso demonstrated a high correlation ofHPV DNA detection,both high-risk andlow-risk types,in self-collected swabscompared with swabs collected byhealth care professionals.31-35There arefew studies comparing correlation ofspecific HPV types.Available studies ontype-specific correlations suggest dif-ferent types may be detected from thesespecimens,although a strong correla-tion,at least in1study,existed for HPVtypes6,11,16,and18.35There are noevaluations in the general US popula-tion using self-collected swabs preclud-ing a direct comparison of our data withthose from other studies.There were several limitations toour study.Nonresponders were sig-nificantly different from respondersby certain demographic characteris-tics(race,age,country of birth,andever had sex)and this could biasprevalence estimates;however,asnoted,evaluations of the weightedprevalence estimates taking the non-responders into consideration didnot substantially change our pointestimates.A self-collected cervico-vaginal sample was obtained,whichmay not detect the same HPV typesas cervical mucosa samples obtainedby health care professionals.Also,HPV DNA point prevalence will mostcertainly underestimate cumulativeincidence as many infections clear9,10;this assessment only measures cur-rent infection and does not indicatepast exposure to HPV.A study in aprevious NHANES sample foundoverall HPV-16seroprevalence infemales to be17.9%,with the preva-lence peaking in20-to29-year-olds.36Seroprevalence provides a bet-ter estimate of cumulative exposure.The difference between the seroprev-alence found in that study and thecurrent DNA prevalence in our studyreflects the high clearance of HPVinfections.This assessment,as allTable3.Multivariate Analysis of Factors Associated With HPV Infection Among Sexually Active Females Aged18to59Years,NHANES2003-2004*CharacteristicOdds Ratio(95%Confidence Interval)PValue†Age,y18-19 1.41(0.78-2.55)20-24 2.20(1.33-3.62).0125-290.81(0.54-1.23)30-591[Referent]Marital statusMarried1[Referent]Widowed,divorced,separated 3.09(1.79-5.34).003Never married 2.20(1.39-3.49)Living with partner 3.37(1.92-5.91)No.of lifetime sex partners11[Referent]2 2.07(0.98-4.38).01Ն3 2.74(1.52-4.94)Total No.of sex partners in past year‡01[Referent]1 2.05(1.04-4.06).022 4.04(1.79-9.11)Ն3 4.12(1.72-9.85)Abbreviations:HPV,human papillomavirus;NHANES,National Health and Nutrition Examination Survey.*Includes1288participants who were18to59years,reported ever having sex,and who had complete information.Goodness of fit based on Hosmer-Lemeshow Satterthwaite adjusted F5=2.0,P=.13.†By Hosmer-Lemeshow Satterthwaite adjusted F test.‡Question was asked only to those females older than18years.HPV PREVALENCE©2007American Medical Association.All rights reserved.assessments of HPV DNA,could not determine if the HPV detected was from the participant or a partner,or if it represented active infection. Finally,we had only2years of data; the distribution of HPV types may change with additional years of data and a larger sample.We did not pre-sent demographic and behavioral fac-tors associated with HPV vaccine type infection because the analysis was limited to subgroups with preva-lence estimates in which the relative SE was30%or less;a relative SE of more than30%means that the SE is quite large relative to the estimate and hence considered unreliable.CONCLUSIONOur study provides the first national es-timate of prevalent HPV infectionamong females aged14to59years inthe United States.Overall,HPV preva-lence was high(26.8%),and preva-lence was highest among females aged20to24years.Our data indicate thatthe burden of prevalent HPV infectionamong women was higher than previ-ous estimates.However,the preva-lence of HPV vaccine types was rela-tively low.Author Contributions:Dr Dunne had full access to allof the data in the study and takes responsibility forthe integrity of the data and the accuracy of the dataanalysis.Study concept and design:Unger,McQuillan,Markowitz.Acquisition of data:Unger,McQuillan,Swan,Patel,Markowitz.Analysis and interpretation of data:Dunne,Unger,Sternberg,McQuillan,Patel,Markowitz.Drafting of the manuscript:Dunne,Patel,Markowitz.Critical revision of the manuscript for important intel-lectual content:Dunne,Unger,Sternberg,McQuillan,Swan.Statistical analysis:Sternberg,McQuillan.Administrative,technical,or material support:Unger,McQuillan,Swan.Study supervision:Unger,Markowitz.Financial Disclosures:None reported.Funding/Support:This study was supported by the Di-vision of STD Prevention,Centers for Disease Con-trol and Prevention.Role of the Sponsor:The funding organization,Na-tional Center for Health Statistics,Centers for Dis-ease Control and Prevention,assisted with the con-duct of the study,in the collection and managementof the data,and in the preparation and review of themanuscript.REFERENCES1.Weinstock H,Berman S,Cates W.Sexually trans-mitted infections in American youth:incidence and prevalence estimates,2000.Perspect Sex Re-prod Health.2004;36:6-10.2.Revzina NV,Diclemente RJ.Prevalence and inci-dence of human papillomavirus infection in women in the USA:a systematic review.Int J STD AIDS.2005; 16:528-537.3.Tarkowski TA,Koumans EH,Sawyer M,et al.Epi-demiology of human papillomavirus infection and ab-normal cytologic test results in an urban adolescent population.J Infect Dis.2004;189:46-50.4.Winer RL,Lee SK,Hughes JP,Adam DE,Kiviat NB, Koutsky LA.Genital human papillomavirus:infection incidence and risk factors in a cohort of female uni-versity students.Am J Epidemiol.2003;157:218-226.5.Trottier H,Franco EL.The epidemiology of genital human papillomavirus infection.Vaccine.2006; 24(suppl1):S1-S15.6.Manhart LE,Holmes KK,Koutsky LA,et al.Hu-man papillomavirus infection among sexually active young women in the United States:implications for developing a vaccination strategy.Sex Transm Dis. 2006;33:502-508.7.Walboomers JM,Jacobs MV,Manos MM,et al. Human papillomavirus is a necessary cause of inva-sive cervical cancer worldwide.J Pathol.1999;189:12-19.8.Bosch FX,de Sanjose S.Chapter1:human papil-lomavirus and cervical cancer-burden and assess-ment of causality.J Natl Cancer Inst Monogr.2003;(31):3-13.9.Moscicki AB,Shiboski S,Broering J,et al.The natu-ral history of human papillomavirus infection as mea-sured by repeated DNA testing in adolescent and young women.J Pediatr.1998;132:277-284.10.Franco EL,Villa LL,Sobrinho JP,et al.Epidemi-ology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer.J Infect Dis.1999;180:1415-1423.11.Food and Drug Administration.Gardasil(quadri-valent human papillomavirus[types6,11,16,18]re-combinant vaccine)[package insert].Whitehouse Sta-tion,NJ:Merck&Co;2006. /cber/label/HPVmer060806LB.pdf.Accessed February 8,2007.12.Koutsky LA,Ault KA,Wheeler CM,et al;Proof of Principle Study Investigators.A controlled trial of a human papillomavirus type16vaccine.N Engl J Med.2002;347:1645-1651.13.Villa LL,Costa RL,Petta CA,et al.Prophylacticquadrivalent human papillomavirus(types6,11,16,and18)L1virus-like particle vaccine in young wom-en:a randomised double-blind placebo-controlled mul-ticentre phase II efficacy ncet Oncol.2005;6:271-278. Centers for Disease Control and Prevention.ACIP provisional recommendations for the use ofquadrivalent HPV vaccine./nip/recs/provisional_recs/hpv.pdf.Accessed February8,2007.15.National Center for Health Statistics.NHANES2003-2004./nchs/about/major/nhanes/nhanes.Accessed February23,2006. Census Bureau.Ratio of income to povertylevel./hhes/income/defs/ratio.html.Accessed April27,2006. Census Bureau Web site.National estimates:monthly population estimates,2000to2004.http:///popest/national/asrh/2004_nat_detail.html.Accessibility verified January25,2007.18.Gravitt PE,Peyton CL,Apple RJ,Wheeler CM.Genotyping of27human papillomavirus types by usingL1consensus PCR products by a single-hybridiza-tion,reverse line blot detection method.J ClinMicrobiol.1998;36:3020-3027.19.Gravitt PE,Peyton CL,Alessi TQ,et al.Improvedamplification of genital human papillomaviruses.J ClinMicrobiol.2000;38:357-361.20.Peyton CL,Gravitt PE,Hunt WC,et al.Determi-nants of genital human papillomavirus detectionin a US population.J Infect Dis.2001;183:1554-1564.21.Vernon SD,Unger ER,Williams parisonof human papillomavirus detection and typing by cyclesequencing,line blotting,and hybrid capture.J ClinMicrobiol.2000;38:651-655.22.SAS Institute Inc.SAS Companion for the Micro-soft Window Environment,Version9.Cary,NC:SASInstitute Inc;2002.23.Shah BV,Barnwell BG,Bieler GS.SUDAAN User’sManual:Release9.0.Research Triangle Park,NC:Re-search Triangle Institute;2004.24.Korn E,Graubard B.Analysis of Health Surveys.New York,NY:Wiley;1999.25.National Center for Health Statistics.Design andestimation for the National Health Interview Survey,1995-2004.Vital Health Stat2.2000;130:1-31.26.Casella G,Berger R.Statistical Inference.PacificGrove,Calif:Wadsworth&Brooks/Col;1990.27.Xu F,Sternberg MR,Kottiri BJ,et al.Trends in her-pes simplex virus type1and type2seroprevalence inthe United States.JAMA.2006;296:964-973.28.Castle PE,Schiffman M,Bratti MC,et al.A popu-lation-based study of vaginal human papillomavirusinfection in hysterectomized women.J Infect Dis.2004;190:458-467.29.Dzuba IG,Diaz EY,Allen B,et al.The acceptabil-ity of self-collected samples for HPV testing vs.thepap test as alternatives in cervical cancer screening.J Womens Health Gend Based Med.2002;11:265-275.30.Kahn JA,Bernstein DI,Rosenthal SL,et al.Ac-ceptability of human papillomavirus self testing infemale adolescents.Sex Transm Infect.2005;81:408-414.31.Sellors JW,Lorincz AT,Mahony JB,et -parison of self-collected vaginal,vulvar and urinesamples with physician-collected cervical samples forhuman papillomavirus testing to detect high-gradesquamous intraepithelial lesions.CMAJ.2000;163:513-518.32.Gravitt PE,Lacey JV Jr,Brinton LA,et al.Evalu-ation of self-collected cervicovaginal cell samples forhuman papillomavirus testing by polymerase chainreaction.Cancer Epidemiol Biomarkers Prev.2001;10:95-100.33.Ogilvie GS,Patrick DM,Schulzer M,et al.Diag-nostic accuracy of self collected vaginal specimensfor human papillomavirus compared to cliniciancollected human papillomavirus specimens:ameta-analysis.Sex Transm Infect.2005;81:207-212.34.Lorenzato FR,Singer A,Ho L,et al.Human pap-illomavirus detection for cervical cancer prevention withpolymerase chain reaction in self-collected samples.Am J Obstet Gynecol.2002;186:962-968.35.Baldwin S,Santos C,Mendez Brown E,et -parison of type-specific human papillomavirus datafrom self and clinician directed sampling.GynecolOncol.2005;97:612-617.36.Stone KM,Karem KL,Sternberg MR,et al.Sero-prevalence of human papillomavirus type16infec-tion in the United States.J Infect Dis.2002;186:1396-1402.HPV PREVALENCE©2007American Medical Association.All rights reserved.。

线粒体DNA的提取方法.

protocol
线粒体DNA的提取方法
Protocol for Extracting Mitochondria DNA
实验目的从植物组织提取高纯度线粒体 DNA
实验原理
1、4000rpm 离心可以沉淀分离出叶绿体、细胞核和组织碎片，12, 000 rpm离心才可沉淀分离线粒体。 2、用蔗糖浓度梯度纯化线粒体，不同浓度蔗糖的缓冲液因比重不同而分层。
3、SDS可以从植物各样组织类型中释放和结合的细胞核酸。
仪器设备： Apparatus:
研钵和研杵；冷冻离心机；过滤装置（漏斗，玻棒） pH 计；移液器；水浴箱； 50mL 离心管；2；1.5 mL离心管。 mortar (cold); Centrifuged; filter; pH meter; water bath boiler; 50mL centrifugal tube; eppendorf tube .
5.弃上清，沉淀中加入0.1 mL/g FW 的缓冲液A，离心管底部再缓缓加入 0.2 mL/g FW 的缓冲液 B ， 12 000rpm离心20 min，所得沉淀即为纯化的线粒体 Re-suspend the organelle pellet with a soft paintbrus in minimal volume of A buffer (0. 1 mL/g FW), B-buffer were added slowly to the tube bottom making a final concentration 0.2mL/g FW of material again. Collect the organelle pellets by centrifugation, 12,000rpm, for 20 min. Keep the pellet at -20℃ or lyse it at once Keep the pellet at -20℃ or lyse it at once

WATSON PHASE DIVIDER GEL 分层凝胶产品说明书

PHASE DIVIDER GELLIGHT AND HEAVYProduct Number Tube Size Sample SizeP 2098 15 ml 1-6 mlP 2348 50 ml 5-20 ml LightP 2598 3 ml syringe CustomP 2223 15 ml 1-6 ml HeavyP 2723 3 ml syringe CustomTECHNICAL BULLETINProduct DescriptionAfter organic extraction, it is often difficult to recover nucleic acid in the aqueous upper phase free from the denatured protein at the aqueous and organic phase interface. Phase Divider Gel, when used during phenol, phenol:chloroform, or phenol:chloroform:isoamyl alcohol extractions, migrates under centrifugal force to form a seal between the organic and aqueous phases. The organic phase and the interface material are effectively trapped below the Phase Divider Gel. The barrier is sufficiently durable that the aqueous upper phase, containing the nucleic acid, can then be recovered quantitatively by simply decanting or pipetting to a fresh tube.Phase Divider Gel is inert, stable to heating, and does not interfere with standard nucleic acid restriction and modification enzymes. In fact, many of the reactions can be carried out in the presence of Phase Divider Gel at the appropriate temperature and then terminated by the addition of phenol or phenol:chloroform. The nucleic acid can then be extracted by following standard protocols. Phase Divider Gel does not interfere with the heat inactivation of enzymes (65 °C for 10 minutes) prior to organic extraction. Note that Phase Divider Gel is not suitable for use with TriReagent® (Product Number T 9424). Phase Div ider Light Gel can be used to improve the recovery of DNA from low melting point agarose with only minor changes to the standard protocol. It may also be used in the standard protocols for the preparation of plasmid DNA from E. coli, phagemid DNA from M13-type phage, and phage DNA from lambda. Phase Divider Light Gel also may by used for isolating high molecular weight genomic DNA from blood, cultured cells, and tissue. The use of Phase Divider Heavy Gel in a microprep plasmid DNA purification protocol allowed for the direct screening of inserts as well as probe synthesis and/or DNA sequencing within one day. Phase Divider Heavy Gel may be used to prepare partially purified plasmid DNA from E. coli and for the preparation of total RNA by homogenization in guanidine thiocyanate and organic extraction.The ability of Phase Divider Gel to separate the phases is dependent upon the composition of the aqueous and the organic media. Salt and protein in the aqueous phase have an effect on both the aqueous and organic phase densities, while different organic phase formulations also vary in density. For optimum phase separation, the compositions of the aqueous phase, organic phase, and Phase Divider Gel must be compatible. As a result, Phase Divider Gel is offered in two different density formulations, Light and Heavy. Pleas consult the following table for the formulation, which fits the desired application.Precautions and DisclaimerPhase Divider Gels are for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Storage/StabilityStore Phase Divider Gels at room temperature.Do not freeze.Applications and CompatibilityChoosing the correct density of Phase Divider Gel for a system is crucial. Consult the table below to select the correct density for the specific application.Organic phaseAqueous phase Phenol:Chloroform:Isoamylalcohol (25:24:1)Chloroform:Isoamylalcohol (24:1)Water or Buffer-saturatedPhenol/Chloroform(1:1)Water or Buffer-saturatedPhenol≤0.5 M NaCl Light, Heavy Light, Heavy Light, Heavy Light≤1 mg/ml BSA Light, Heavy Light, Heavy Light, Heavy LightCleared lysate Heavy Heavy Heavy Not compatible* HomogenizedtissueLight, Heavy Light, Heavy Light, Heavy LightRNA purification Heavy Heavy Heavy Not compatible* *This combination for aqueous and organic phases is not suitable for use with Phase Divider Gel.Note that Phase Divider Gel is not suitable for use with Tri Reagent.ProcedureNote: for optimum results with Phase Divider Light Gel, the starting sample should not exceed 0.5 M NaCl or1 mg/ml protein. Samples exceeding these concentrations should be diluted prior to extraction. If dilution is inappropriate, extractions may be performed with Phase Divider Heavy Gel in combination with phenol:chloroform:isoamyl alcohol (25:24:1),phenol:chloroform (1:1), or chloroform:isoamyl alcohol (24:1). Phase Divider Heavy gel is not compatible with Water or buffer-saturated phenol as shown in the table.Note: Phase Divider tubes should be centrifuged immediately prior to adding the aqueous and/or organic phases to them.A. Phase Divider Gel in 15 and 50 ml tubes: GeneralProtocolImmediately prior to use, centrifuge the Phase Divider Gel at 1500 X g for 1-2 minutes. 1. Add 1-6 ml (15) or 5-20 ml (50) of aqueous sampleand an equal volume of organic extraction solvent directly to the centrifuge tube containing PhaseDivider Gel.2. Thoroughly mix the organic and aqueous phasesby repeated inversion to form a transientlyhomogeneous suspension. The Phase Divider Gel will not become part of the suspension. DO NOTVORTEX.3. Centrifuge for two minutes at 1500 X g full speed toseparate the aqueous and organic phases. ThePhase Divider Gel will form a barrier between theaqueous and organic phases. A small amount ofPhase Divider Gel may remain in the bottom of the tube.4. Carefully decant, or transfer by pipette, theaqueous upper phase containing the nucleic acid toa fresh tube.5. Precipitate the nucleic acid by adding salt, alcohol,and carrier (if needed) as specified in your protocol or application.B. Phase Divider in Syringe: General Protocol1. Without putting pressure on the plunger, twist offthe orange cap and discard. Screw on the supplied gray dispensing tip to the syringe and tightensecurely.2. Apply firm pressure on the plunger to dispense thePhase Divider Gel.3. The following amounts of Phase Divider Gel arerecommended for optimum phase separation.a. Approximately 100 µl Phase Divider Gel for0.65 ml microcentrifuge tubes.b. Approximately 300 µl Phase Divider Gel for 1.5ml microcentrifuge tubes.c. Approximately 2 ml Phase Divider Gel fordisposable 15-ml screw-cap centrifuge tubes.d. Approximately 5 ml Phase Divider Gel fordisposable 50-ml screw-cap centrifuge tubes.4. Store the assembled syringe by pulling back on theplunger slightly. It is recommended that the gray tip be left in place, as Phase divider Gel is quiteslippery and can prevent adequate securing of the tip in the future.C. Recovery of DNA from Low Melting Point Agarose1. Resolve the DNA fragments on a low melting pointagarose (Product Number A 9414) in 1X Tris-Acetate-EDTA (TAE) buffer. Do not use TBEbuffer as borate gels are much more difficult to dissolve.2. Stain the gel with ethidium bromide. Visualize witha long wave UV light and carefully cut out theband(s) of interest with a sharp razor blade. (Note: Wear gloves when handling ethidium bromide and stained gels.)3. Transfer the gel section to a pre-spun (12,000 X gfor 20-30 seconds), pre-weighed Phase DividerLight Gel in a 1.5 ml tube and determine the weight of the slice. 4. Add a volume (in µl) of 1X TE, pH 8.0 (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, Product Number T 9285, diluted to working concentration), equivalent to 5Xthe weight (in mg) of the slice. Melt the slice in theTE at 65 °C for 5-10 minutes.5. Mix well to ensure the agarose slice is fully melted.Allow the melted sample to come to roomtemperature, and add an equal volume of roomtemperature buffer-saturated phenol, pH 8.0(Product Number P 4557), to the sample.Thoroughly mix the organic and aqueous phasesby repeated inversion to form a transientlyhomogeneous suspension. The Phase Divider Gel will not become part of the aqueous suspension.DO NOT VORTEX.6. Centrifuge at full speed (12,000 X g or greater in amicrocentrifuge) for two minutes to separate thephases.Note: If the resulting aqueous phase still appears cloudy, the extraction with room temperature buffer-saturated phenol, pH 8.0, may be repeated.7. Transfer the aqueous phase to fresh Phase DividerLight Gel in a 1.5 ml tube and extract with anequivalent volume of room temperature phenol-chloroform-isoamyl alcohol (PCI, 25:24:1, ProductNumber p 3803 or P 2069). DO NOT VORTEX.8. Centrifuge at full speed (12,000 X g or greater in amicrocentrifuge) for two minutes to separate thephases. Transfer the aqueous phase to freshPhase Divider Light Gel in a 1.5 ml tube and extract with an equivalent volume of room temperaturechloroform-isoamyl alcohol (CI, 24:1, ProductNumber C 0549).9. Centrifuge at full speed (12,000 X g or greater in amicrocentrifuge) for two minutes to separate thephases and transfer the aqueous phase to asuitably sized microcentrifuge tube.10. Add 0.25 volume of 10 M ammonium acetate and2.5 volumes of 100% ethanol to the sample andmix well.11. Incubate at room temperature for 20 minutes,centrifuge to form pellet, wash the pellet severaltimes with cold 70% ethanol, air-dry the pellet, and resuspend in a suitable buffer.References1. Birnboim, H. C., A rapid alkaline extractionprocedure for screening recombinant plasmid DNA.Nucleic Acids Res., 7, 1513-1523 (1979).2. Birnboim, H. C., Rapid extraction of high molecularweight RNA from cultured cells and granulocytes of Northern analysis. Nucleic Acids Res., 16, 1487-1497 (1988).3. Chirgwin, J. M., et al., Isolation of biologically activeribonucleic acid from sources enriched inribonucleases. Biochemistry, 18, 5294-5299 (1979).4. Chomczynski, P., and Sacchi, N., Single-stepmethod of RNA isolation by acid guanidiniumthiocyanate phenol-chloroform extraction. Anal.Biochem., 162, 159-159 (1987).5. Current Protocols in Molecular Biology, Ausubel, F.M., et al., (Eds.), pp 1.14.1-1.15.4. (John Wiley and Sons, New York, NY, 1989). 6. Emanuel, J.. R., Phase Lock Gel plasmidmicropreps: direct insert screening, probesysnthesis, and sequencing within one day. Nucleic Acids Res., 20, 625 (1992).7. Laws, G. M., and Adams, S. P., Measurement of 8-OhdG in DNA by HPLC/ECD: the importance ofDNA purity. BioTechniques, 20, 36-38 (1996).8. Murphy, N. R., and Hellwig, R. J., Improved nucleicacid organic extraction through use of a unique gel barrier material. BioTechniques, 21, 394 (1996). 9. Molecular Cloning: A Laboratory Manual, ThirdEdition. Sambrook, J., and Russell, D. W.,(Eds.)(Cold Spring Harbor Laboratory Press, NewYork, 2001). (Product Code M 8265).JWM 02/05Tri Reagent is a Registered Trademark of Molecular Research CenterSigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。

蔗糖梯度离心

Sucrose Gradient SeparationProtocolCONTENTI. R EQUIRED R EAGENTS AND E QUIPMENT3 II. S AMPLE P REPARATION 4 III. S AMPLE S OLUBILIZATION5 IV. S UCROSE G RADIENT P REPARATION6 V. C ENTRIFUGATION OF G RADIENTS AND E LUCTION OF F RACTIONS8 VI. S AMPLE A NALYSIS9 VII. B UFFER R ECIPES10 VIII. O PTIMIZATION S TEPS AND G ENERAL T IPS11 IX. T ROUBLESHOOTING G UIDE13 X. F LOW C HART14 XI. N OTES15I. REQUIRED REAGENTS AND EQUIPMENTReagents:•n-dodecyl- -D-maltopyranoside (Lauryl maltoside; MitoSciences MS910)Phosphate buffered saline, PBS (section VII)Sucrose solutions 15, 20, 25, 27.5, 30 and 35 %Double distilled waterProtease inhibitor cocktail (section VII)13 x 51 mm polyallomer centrifuge tubes (Beckman 326819) Equipment:Swing-out compatible ultracentrifuge and rotor (e.g. Beckman SW50.1)pH meter, weighing balance and other standard lab equipment Laboratory benchtop microfugeProtein electrophoresis and Western blotting equipmentII. SAMPLE PREPARATIONThe MitoSciences sucrose gradient separation procedure is a protein subfractionation method optimized for mitochondria. It can be used to reduce sample complexity facilitating large-scale proteomics efforts (Taylor et al, 1Hanson et al). Also when this procedure was coupled to MitoSciences highly sensitive Western blotting antibodies it allows the detection of mis-assembled mitochondrial enzyme complexes in patient cell lines (2Hanson et al).This method resolves a sample into at least 10 fractions. It is possible to separate solubilized whole cells into fractions of much lower complexity but when analyzing already isolated mitochondria the fractions are even more simplified. Details of mitochondrial isolation can be found at /PDF/mitos.pdfThe total amount of OXPHOS complexes in mitochondrial samples/ cell samples varies greatly between species and tissues. Therefore it is highly recommended that, during the experimental planning steps, an estimation of the total amount of protein in the users sample is made. In this way the appropriate detection strategy can be employed. Table I suggests detection strategies based amount of starting material.Table 1. Suggested detection methods by amount starting material.Starting mitochondria applied to the gradient Recommended detection strategy5 mg + Gel staining with coomassie0.5 mg + Gel staining with silver/sypro ruby< 0.5 mg Western blotting with MitoSciences mAbs Taylor SW, Fahy E, Zhang B, Glenn GM, Warnock DE, Wiley S, Murphy AN, Gaucher SP, Capaldi RA, Gibson BW, Ghosh SS. Characterization of the human heart mitochondrial proteome. Nat Biotechnol. 2003 Mar; 21(3): 239-40.1Hanson BJ, Schulenberg B, Patton WF, Capaldi RA. A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map. Electrophoresis. 2001 Mar; 22(5): 950-9.2Hanson BJ, Carrozzo R, Piemonte F, Tessa A, Robinson BH, Capaldi RA. Cytochrome c oxidase-deficient patients have distinct subunit assembly profiles. J Biol Chem. 2001 May 11; 276(19): 16296-301.III. SAMPLE SOLUBILIZATIONThe sucrose gradient separation technique detailed in this manual is designed for an initial sample volume of up to 0.5 ml at 5 mg/ml protein. Therefore 2.5 mg or less of total protein should be used. For larger amounts, multiple gradients can be prepared or larger scale gradients are also possible, see section VIII.The sample should be solubilized in a non-ionic detergent. It has been determined that at this protein concentration mitochondria are completely solubilized by 20 mM n-dodecyl- -D-maltopyranoside (1% w/v lauryl maltoside). The key to this solubilization process is that the membranes are disrupted while the previously membrane embedded multisubunit OXPHOS complexes remain intact, a step necessary for this density based sucrose separation procedure described here*.To a mitochondrial membrane suspension at 5 mg/ml protein in PBS add lauryl maltoside to a final concentration of 1 %. Mix well and incubate on ice for 30 minutes. Centrifuge at 72 000 g for 30 minutes. The Beckman Optima benchtop ultracentrifuge is recommended for small sample volumes** (however at a minimum a benchtop microfuge on maximum speed should suffice, which is usually around 16 000 g). The supernatant is collected and the pellet discarded. Add a protease inhibitor cocktail (see section VII) and keep the sample on ice until centrifugation is performed. Note: with samples very rich in mitochondria the cytochromes in complexes III and IV may give this supernatant a brown color, which is useful when checking the effectiveness of the separation in section V.* One important exception is the pyruvate dehydrogenase enzyme: In order to isolate PDH at a protein concentration of 5 mg/ml mitochondria, the required detergent concentration is only 10 mM (0.5 %) lauryl maltoside. ** The PDH enzyme should also be centrifuged at lower speeds, a centrifugal force of 16 000 g is maximum for the PDH complex.IV. SUCROSE GRADIENT PREPARTIONA discontinuous sucrose density gradient is prepared by layering successive decreasing sucrose densities solutions upon one another. The preparation and centrifugation of a discontinuous gradient containing sucrose solutions from 15-35 % is described in detail in this manual. This gradient gives good separation of the mitochondrial OXPHOS complexes (masses ranging from 200 kDa to 1000 kDa). However this setup can be modified for the separation of a particular complex or for the separation of larger amounts of material (the details of optimization steps and considerations are given in section VIII).Sucrose sample 15 %20 %25 %27.5%30 %35 %0.5 ml1 ml 1 ml 1 ml 0.75 ml 0.5 ml 0.25 mlallow to run down inside of tube15-35 % sucrose density gradient . The preparation of these sucrose solutions is described in section VII. The gradient is prepared by layering progressively less dense sucrose solutions upon one another; therefore the first solution applied is the 35 % sucrose solution. A steady application of the solutions yields the most reproducible gradient therefore the setup described above in Figure 1 is recommended. Firstly a Beckman polyallomer tube is held upright in a tube stand. Next a yellow (200 μl) pipettor tip is placed on the end of a blue (1000 μl) pipettor tip. Both snugly fitting tips are held steady by a clamp stand and the end of the yellow tip is allowed to make contact with the inside wall of the tube as shown below. Now sucroseFigure 1. Setup for sucrose gradient preparationsolutions can be placed inside the blue tip and gravity will feed thesolutions into the tube slowly and steadily, starting with the 35 %solution (the volumes of this and the other solutions are shown in Table II). Note: if the solutions fail to feed down through the tips andinto the tube a small amount of positive air pressure can be applied tothe blue tip to start the flow. This is done by gently tapping on thewide end of the blue tip. Once the 35 % solution has drained into the tube, the 30 % solution can be loaded into the blue tip which will then flow down the inside of the tube and layer on top of the 35 % solution. This procedure is continued with the 27.5 %, 25 %, 20 % and 15 % respectively. There should now be enough space left at the top of the tube upon which to pipette the 0.5 ml sample of solubilized mitochondria described in section III.Solution Volume1 (top) Sample 0.5 ml2 15 % sucrose 1 ml3 20 % sucrose 1 ml4 25 % sucrose 1 ml5 27. 5 % sucrose 0.75 ml6 30 % sucrose 0.5 ml7 (bottom) 35 % sucrose 0.25 ml5 ml totalTable II. Volumes of solutions used.V. CENTRIFUGATION AND ELUTION OF FRACTIONSOnce the sucrose gradient is poured discrete layers of sucrose should be visible. Having applied the sample to the top of the gradient the tube should be handled and loaded into the rotor very carefully. Centrifugation should begin as soon as possible. However all centrifugation procedures require a balanced rotor therefore another tube containing precisely the same mass must be generated. In practice this means 2 gradients must be prepared although the second gradient need not contain an experimental sample but could contain 0.5 ml water in place of the 0.5 ml protein sample.The polyallomer tubes should be centrifuged in a swinging bucket SW 50.1 type rotor (Beckman) at 37 500 rpm (RCF av 132 000 x g) for 16 hours 30 minutes at 4 o C with an acceleration profile of 7 and deceleration profile of 7.Immediately after the run the tube should be removed from the rotor, taking great care not to disturb the layers of sucrose. When separating a sample rich in mitochondria, discrete colored protein layers might be observable. Most often these are Complex III (500 kDa – brown color) approximately 10 mm from the bottom of the tube and Complex IV (200 kDa – green color) 25 mm from the bottom of the tube. In some circumstances additional bands can be observed. These are the other OXPHOS complexes.For fraction collection the tube should be held steady and upright by a clamp stand. A tiny hole should be introduced into the very bottom of the tube using a fine needle. The hole should be just big enough to allow the sucrose solution to drip out at approximately 1 drop per second. Fractions of equal volume are then collected in eppendorf tubes below the pierced hole. A total of 10 x 0.5 ml fractions are appropriate however collecting more fractions which are thus smaller in volume is also possible e.g. 20 x 0.25 ml fractions. The fractions can now be stored at – 80o CV. SAMPLE ANALYSISFractions collected can now be resolved by electrophoresis. Resolved proteins should be detected by the method chosen in Table I above. Optimized protocols for electrophoresis, gel staining and Western blotting can be found at /PDF/western.pdfAs described in these protocols samples are first solubilized in SDS-PAGE sample buffer, see section VII. Prior to loading the sample can be reduced by 50 mM DTT or 1 % -mercaptoethanol. Another optional step is heating of the sample, which can be done at 95o C for 5 minutes or at 37o C for 30 minutes prior to loading onto the gel. These steps may increase the resolution of protein bands and also reduce the complexity of the sample by breaking any disulfide bonded proteins.A typical example of fractions taken from a sucrose gradient of solubilized heart mitochondria are shown below. 10 μl of each fraction was resolved by SDS-PAGE before coomassie staining.1 2 3 4 5 6 7 8 9 10Figure 2. A typical sucrose gradient separation of mitochondriashowing enrichment of OXPHOS Complex I (predominates in lane 2), Complex II (lane 8), Complex III (lane 4 & 5), Complex IV (lane 7 and 8), and Complex V (lane 4).VI. BUFFER RECIPESPhosphate buffered saline solution (PBS)1.4 mM KH2PO48 mM Na2HPO4140 mM NaCl2.7 mM KCl, pH 7.3------------------------------------------------------------------------------- Lauryl maltoside stock200 mM n-dodecyl- -D-maltopyranoside (10 % w/v lauryl maltoside) (Anatrace D310S)------------------------------------------------------------------------------- Protease inhibitor stocks (each is 1000 x)1 M phenylmethanesulfonyl fluoreide (PMSF) in acetone (Sigma L7626) 1 mg/ml leupeptin (Sigma L2884)1 mg/ml pepstatin (Sigma P4265)------------------------------------------------------------------------------- Sucrose solutionsTo six 15 ml tubes polypropylene centrifuge tubes (Corning)add 1.5g, 2g, 2.5g, 2.75g, 3g and 3.5 g sucrose.Fill each tube to 10 ml with 50 mM Tris. HCl pH 7.5, 1 mM EDTA,0.05 % lauryl maltoside.Turn the tubes on a rotator for approximately 20 minutes until all the sucrose has dissolved.------------------------------------------------------------------------------- 2x SDS page sample buffer20 % glycerol4 % SDS100 mM Tris pH 6.80.002 % Bromophenol blueoptional – 100 mM dithiothreitol-------------------------------------------------------------------------------VII. OPTIMIZATION STEPS AND GENERAL TIPSProtein determinationEach fraction should be 500 μl (or less if greater than 10 fractions were collected). The protein content within each fraction can be determined by protein assay, we recommend the BCA or micro BCA methods (Pierce).Protein precipitation for concentration and/or sucrose removal The proteins in the sample can be precipitated for concentration and sucrose removal however precipitated proteins will be denatured. We recommend the Chloroform/methanol precipitation method. Briefly add 600 μl fraction methanol to a 150 μl of a fraction. After thorough mixing 150 μl chloroform is added. Vortex then add 450 μl water, vortex again (appears cloudy white) and centrifuge immediately for 5 minutes at full speed in a microfuge. A white disc of protein should form between the organic layer (bottom) and the aqueous layer (upper). Discard the upper aqueous layer. Add 650 μl of methanol to the tube and invert 3 times. Spin for 5 minutes at full speed in a microfuge. Remove all liquid and allow the pellet to air dry. The precipitated protein pellet can be taken up in SDS PAGE sample buffer for electrophoresis and/or protein assay.Other sucrose gradient formulationThe sucrose gradient concentrations and number of layers can be altered when optimizing for the separation of a particular enzyme complex. For example to improve the separation of Complex I (the largest complex) proportional volume of the 27.5 %, 30 % and 35 % sucrose solutions should be increased. An example of such a gradient would be 500 μl each of a 37.5% 35%, 32.5%, 30%, 27.5%, 22.5%, 20%, 17.5%, and 15% sucrose solution and a 500 μl sample makes the total volume of 5 ml.Larger sucrose gradients are possible– an exampleGradients larger than 5 ml can be prepared for separation of starting samples greater than 2.5 mg protein (i.e. 500 μl sample at 5 mg/ml). However larger tubes and centrifuge rotors are necessary.As an example 40 mg of mitochondria can be resolved by a 55-25%41 ml sucrose gradient. This is made by layering sucrose solutions of55% (5 ml), 50% (5 ml), 35% (10 ml), 30% (10 ml), 25% (3 ml), and finally on top 8 ml of solubilized mitochondria (which is thesupernatant from 8 ml of mitochondria (5 mg/ml) solubilized by 1%lauryl maltoside and centrifuged 30 min 72 000 g as described abovein section III). This gradient should be centrifuged at 37 000 rpm (113 000 RCF av) in a VTi50 type rotor for 17 hours at 4o C with acceleration profile 9 and deceleration profile 9.VIII. TROUBLESHOOTING GUIDENo bands appear after centrifugation In order to see resolved bands thesample must be rich in mitochondria.Prior to centrifugation the sample mightappear colored brown. If not then themitochondrial content may be lower andtherefore the more sensitive methods ofgel staining/Western blotting detectionshould be chosen.The bands may have diffused aftercentrifugation before fraction collectionThe sucrose layers may have beendisturbedWeak or no gel staining signal Isolate mitochondria from the sampleIncrease the amount of sampleSilver stain the gelWestern blot the gel and detect withMitoSciences antibodiesIsolate the mitochondria to higher purity Non-specific bands when WesternblottingAdd a reducing agent to the elutedsample e.g. DTTHeat the eluted sample at 95 o C for 5minutes before loadingIX. FLOW CHARTThis guide is for quick reference only. Be completely familiar with the previous details of this document before performing the assay.2.5 mg mitochondria in 0.45 ml PBS, supplemented with1 μg/ml pepstatin, 1μg/ml leupeptin, 1mM PMSFAdd 50 μl 10% LM incubate on ice 30 minutesCentrifuge 72 000g 4o C 10 minutesPrepare the following sucrose solutions in Buffer A35% (0.25 ml), 30% (0. 5 ml), 27.5% (0.75 ml),25% (1 ml), 20% (1ml)Load the 0.5 ml solubilized mitochondria on top ofthe prepared sucrose gradientCentrifuge SW 50.1 for 16.5 hours at 37 500 rpmPierce the bottom of the tube and collect10 (or more) fractions of equal volume.Perform protein assay and electrophoresis/Western blotting.Buffer A : 50 mM Tris. HCl pH 7.5Leupeptin : 1 mg/ml (water)Pepstatin : 1 mg/ml (ethanol)PMSF : 0.3 M (ethanol)LM : n-dodecyl- -D-maltosideX. NOTES Phone: 1 800 910-6486 Fax: (541) 284-1801。

dna的甲基化修饰基础实验方案

dna的甲基化修饰基础实验方案DNA Methylation Modification Basic Experimental Protocol.Materials:Genomic DNA.Restriction enzymes (e.g., MspI, HpaII)。

DNA methylation detection kit.Spectrophotometer.Gel electrophoresis equipment.Primers for DNA amplification.Procedure:1. Purify Genomic DNA: Extract genomic DNA from thecells or tissue of interest using a standard DNA isolation protocol.2. Digest DNA with Restriction Enzymes: Digest the DNA samples with two restriction enzymes: MspI and HpaII. MspI recognizes the CCGG sequence, which is typically methylated in mammalian genomes. HpaII recognizes the CCGG sequenceonly when it is unmethylated.3. Measure DNA Concentration: Determine the DNA concentration of each sample using a spectrophotometer.This measurement is necessary to ensure equal amounts of DNA are used in subsequent steps.4. Detect DNA Methylation: Use a DNA methylation detection kit to quantify the amount of methylated DNA in each sample. The kit typically involves a chemical reaction that converts methylated cytosines into a detectable signal.5. Amplify Target DNA Region: Perform PCR (polymerase chain reaction) to amplify a specific region of the DNAthat contains the CCGG sites of interest. This will allow for more accurate quantification of the methylation status.6. Analyze PCR Products: Analyze the PCR products by gel electrophoresis to determine the extent of methylation. The presence of methylated DNA will result in a higher molecular weight fragment, while unmethylated DNA will result in a lower molecular weight fragment.7. Interpret Results: Methylated DNA will be detected by the presence of a higher molecular weight band on the gel. The intensity of the band can be used to estimate the extent of methylation.Chinese Response:DNA甲基化修饰基础实验方案。

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