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Cat. # RR114A For Research Use Bacteria Screening PCR Kit Product Manual v201310Da Bacteria Screening PCR Kit Cat. #RR114A v201310Da Table of Contents I. Description............................................................................................. 3 II. Components.......................................................................................... 3 III. Storage..................................................................................................... 4 IV. Materials Required but not Provided........................................... 4 V. Precautions for Operation................................................................ 4 VI. Principle ................................................................................................. 5 VII. Protocol................................................................................................... 6 VIII. Application Examples......................................................................11 IX. References............................................................................................15 X. Related Products................................................................................15 2 URL:http://www.takara-bio.com Cat. #RR114A Bacteria Screening PCR Kit v201310Da I.Description In order to maintain consumer trust in the safety of food products, a high priority has been placed on assuring product quality at each step of the food supply process. PCR is recognized as one of the more useful methods for such food-related quality control applications. PCR is a technique in which minute amounts of DNA can be used as a template for amplifying only a desired DNA fragment. A single cycle of this method consists of three incubation steps; DNA thermal denaturation, primer annealing, and primer extension with DNA polymerase. Amplification by a factor of 1 million can be achieved in relatively short periods of time by repeating these steps. The Bacteria Screening PCR kit uses the 16S rRNA gene as a target and employs the ENT primer for detecting strains in the Enterobacteriaceae family, including E. coli and Salmonella bacteria, and the BS primer for detecting genus Bacillus strains, including Bacillus cereus , and genus Staphylococcus , including Staphylococcus aureus . (Table 1 lists the reactivity characteristics of the various bacterial groups tested.) When used in combination with an enrichment culture, the Bacteria Screening PCR Kit can be used to rapidly detect the presence of even minute numbers of bacteria in samples. II.Components (50 μl x 50 reactions for ENT and BS primers) 1.2X Premix Solution * 1 2.Primer Mix ENT 3.Primer Mix BS 4.Positive Control ENT 5.Positive Control BS 6.dH2O 7.10% Chelex® Solution * 2 2X conc. 5 μM each 5 μM each 250 μlx 10 (100 reactions) 125 μl (50 reactions) 125 μl (50 reactions) 25 μl (10 reactions) 25 μl (10 reactions) 1 mlx 3 12 mlx 2 * 1:2X Premix Solution contains the followings: TaKaRa Ex Taq HS :0.1 units/μl Optimized Buffer :2X conc. (Mg2+ concentration :4 mM) dNTP Mixture :0.4 mM each * 2:Chelex® is registered trademark of Bio-Rad Laboratories. URL:http://www.takara-bio.com 3 Bacteria Screening PCR Kit Cat. #RR114A v201310Da III.Storage 10% Chelex® solution : Stored at 4℃, shipped at room temperature Other components : Stored and shipped at -20℃ IV.Materials Required but not Provided [ Reagents ] -Agarose L03 (Cat. #5003) or NuSieve™ 3:1 Agarose (Lonza; Cat. #50090/50091/50094) -Electrophoresis buffer [ TBE powder (Cat. #T905) , etc. ] -DNA marker ϕX174 Hae III digest (Cat. #3405A/B) 100 bp DNA Ladder (Cat. #3407A/B) , etc. -Loading buffer [ 6X:36% glycerol, 0.05% bromophenol blue, 30 mM EDTA, 0.05% xylene cyanol ] (Note:Supplied with Takara DNA marker.) -DNA Staining Ethidium bromide [ Equipment ] -Thermal cyclers TaKaRa PCR Thermal Cycler Dice Touch (Cat. #TP350)* TaKaRa PCR Thermal Cycler Dice Gradient/Standard (Cat. #TP600/TP650)* -Electrophoresis apparatus -Ultraviolet transilluminator (Wavelength 300 nm) -Polaroid camera or digital imaging system to photograph stained gel -Heating block (applicable at 100℃) -Refrigerated centrifuge, compatible for 1.5 ml tube [ Other ] -0.2 ml PCR tube -Micropipettes and tips (with hydrophobic filter) -Tray for staining agarose gel * : Not available in all geographic locations. Check for availability in your region. V.Precautions for Operation 2X Premix Solution contains enzymes; avoid vigorous mixing. Repeated freezing and thawing will also result in decreased activity. It is advisable to thaw only the required number of tubes of solution, dispense 25-μl aliquots into PCR tubes, and store at -20℃. 4 URL:http://www.takara-bio.com Cat. #RR114A Bacteria Screening PCR Kit v201310Da VI.Principle The PCR (Polymerase Chain Reaction) process consists of the following 3 steps: 1.DNA denaturation step 2.Primer annealing step 3.Polymerase extension step In vitro DNA amplification is achieved by repeating these steps (see figure below) . PCR allows amplification of target DNA by a factor of 1 million in a few hours. 1 Cycle Temperature (℃) Step 1 94 Step 2 Denaturation 35 Cycles Step 3 Step 4 106-fold amplification of target DNA fragment 72 Synthesis of DNA 55 Primer Annealing 22 1 2 3 Time (min.) 4 5 Step 1: Denature the target double-stranded DNA fragment in the reaction mixture containing primers, dNTPs, and polymerase. Step 2: Anneal primer to single-stranded DNA. Step 3: Synthesize DNA with DNA polymerase. Step 4: Return to Step 1 - to denature the amplified double-stranded DNA again to yield single-stranded DNA. Steps 1 to 4 are repeated. Note that the conditions that produce maximum amplification for a given DNA fragment will vary, and must be optimized for each target fragment. URL:http://www.takara-bio.com 5 Bacteria Screening PCR Kit Cat. #RR114A v201310Da VII.Protocol 1.Preparation of Sample Solutions A.DNA Extraction (In cases where large amount of bacteria can be prepared.) 1) Take 20 to 100 μl of bacterial culture media that has been incubated overnight in BHI medium*1, SCD medium*2, or another suitable medium and centrifuge at 12,000 rpm (13,000 X g ) for 3 to 5 minutes. (Use of 1.5 ml microtube with screw cap is recommended.) [ In cases where higher sensitivity is required, wash and suspend the collected bacteria in 1 ml of distilled water or TE buffer (10 mM Tris HCl, 0.1 mM EDTA, pH 8.0) , then centrifuge again. ] 2) Once the bacteria have been separated, add 2 to 10 volumes of pre-mixed 10% Chelex Solution (typically 50 to 200 μl) using a pipet tip (sterile) that has been cut to allow a wider opening to suspend the bacteria. 3) Heat the suspended bacteria solution at 99℃ for 5 minutes, then quickly cool by placing on ice for 1 minute or more. 4) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute. 5) Use the centrifuged supernatant as the DNA sample solution for PCR. B.Preparation of heat-extracted sample directly from bacteria 1) Pick up a minute quantity of the bacteria from the colony and suspend in 50 to 200 μl of 10% Chelex® solution. (Use of a 1.5 ml microtube with screw cap is recommended.) 2) Heat the suspended bacteria solution at 99℃ for 5 minutes, then quickly cool by placing on ice for 1 minute or more. 3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute. 4) Use the centrifuged supernatant as the DNA Sample Solution for PCR. C.Preparation of DNA sample from food sample combined with culturing 1) Suspend the sample to be tested (food sample) 5 to 20 volumes of BHI medium * 1, SCD medium * 2 or another suitable medium, and crush using homogenizer (Stomacher, etc.). Culture with or without agitation for 4 to 7 hours. 2) Centrifuge 1.3 ml of the culture solution at 1,000 rpm (approximately 100 X g ) for 1 minute. Then centrifuge 1.0 to 1.2 ml of the supernatant at 12,000 rpm (approx. 13,000 X g ) for 3 minutes. 3) Add 200 μl of 10% Chelex® solution to the pellet and heat at 99℃ for 5 minutes, then quickly cool by placing on ice for 1 minute or more. 4) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute. 5) Use the centrifuged supernatant as the DNA Sample Solution for PCR. * 1:BHI medium:Brain Heart Infusion medium * 2:SCD medium:Soybean Casein Digest medium 6 URL:http://www.takara-bio.com Bacteria Screening PCR Kit Cat. #RR114A v201310Da Note : In cases where the greater sensitivity is required, use the procedures described in either [ Option 1 ] or [ Option 2 ] below. [ Option 1 ] (Use of a 1.5 ml microtube with screw cap is recommended) 1) Wash the pellet containing the separated bacteria (see step 2 in section C above) using sterile water or TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH8.0) then suspend in a 200 μl of Chelex® solution. 2) Heat at 99℃ for a period of 5 minutes then quickly cool by placing on ice for 1 minute or more. 3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute. 4) Use the supernatant as the DNA Sample Solution for PCR. [ Option 2 ] (Use of a 1.5 ml microtube with screw cap is recommended) 1) Suspend the pellet containing the centrifuged bacteria (see step 2 in section C above) in 100 μl of acromopeptidase*3 (250 U/ml, in TE buffer) and incubate for 10 to 15 minutes at 37 to 55℃. (This enables the enzymes to break down the cell walls of gram-positive bacteria such as Staphylococcus aureus . This, in turn, increases the content of recoverable DNA. Although this process is not required for Enterobacteriaceae and Vibrionaceae bacteria families, it does not adversely affect detection.) 2) Add a 100 μl of Chelex® solution and heat at 99℃ for 5 minutes, then quickly cool by placing on ice for 1 minute or more. 3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute. 4) Use the supernatant as the DNA Sample Solution for PCR. Note 1: Some processed foods (particularly heavily spiced ones) may inhibit PCR. In such cases, purify DNA using a kit such as the Generation Capture Column Kit (QIAGEN) and DNeasy Plant Mini Kit (QIAGEN). Note 2: With food products containing high content of dead bacteria, dead bacteria may be detected, even when combined to culture techniques. Note 3: The Primer Mix BS may react with other bacillus types, such as those found in fermented food products. Note 4: In some cases, Vibionaceae family bacteria may not grow very well in BHI*1 and SCD*3 medium. * 3:Acromopeptidase:Acromopeptidase (TBL-1) and less-refined products URL:http://www.takara-bio.com 7 Cat. #RR114A Bacteria Screening PCR Kit v201310Da 2.Preparation of PCR Reaction Mixture Mix the following components on ice in a 0.2 ml PCR tube. 2X Premix Solution Primer Mix ENT or BS (5 μM each) DNA Sample Solution * 1 dH2O Volume 25.0 μl 2.5 μl 2.5 μl * 2 to 50.0 μl Final conc. 1X 0.25μM each * 1:A mixture containing dH2O instead of DNA sample solution is used as a Negative Control. The positive control mixture is produced by adding 2.5 μl of Positive Control ENT or BS. * 2:DNA sample solution can be added up to 5 μl. Exceeding this volume may result in inhibition of the PCR reactions. 3.PCR Reaction Ensure that the reaction tube cap is firmly tightened, and then load the tube correctly into the Thermal Cycler unit and perform reactions according to the following specifications. 95℃ ↓ 95℃ 59℃ 72℃ ↓ 72℃ 4℃ 60 sec. 30 sec. 30 sec. 30 sec. 30 cycles* 60 sec. *:Perform reactions with 30 cycles. Conducting reactions with an excessive number of cycles can result in secondary amplification products from Taq polymerase. 8 URL:http://www.takara-bio.com Bacteria Screening PCR Kit Cat. #RR114A v201310Da 4.Preparation of Agarose Gel 1) Dispense electrophoresis buffer into a triangular flask, and then add Agarose L03 or NuSieve™ 3:1 Agarose to a concentration of 1.8% (w/v) or 3% (w/v), respectively, with mixing. 2) Heat the mixture for 2 to 3 minutes in a microwave. Remove and mix thoroughly to ensure that the mixture has dissolved uniformly. If not, heat and mix again. 3) Set up the gel tray. 4) Once the agarose gel cools to 50 - 60℃, pour the solution into the tray, insert the comb to generate slots for applying samples, and leave at room temperature for 30 to 60 minutes to harden the gel. * When pre-staining Using Ethidium Bromide. After the agarose gel cools to 50 - 60℃, add ethidium bromide solution to a final concentration of 0.5 μg / ml. Mix thoroughly and pour into the gel tray. Leave at room temperature for 30 - 60 minutes and harden the gel. 5) After the gel hardens, place it in an electrophoresis tank and add electrophoresis buffer so that the gel is completely immersed. 6) Remove the comb, carefully not to break the gel. 5.Electrophoresis 1) Connect the electrophoresis apparatus, taking care to connect the anode and cathode poles correctly. (Note : DNA is negatively charged and will move from cathode to anode.) 2) Add 1.0 μl of 6X loading buffer to 2 to 5 μl of PCR reactant and mix. Gently dispense into the lanes in the gel using a micropippette. (Dispense appropriate amounts of DNA marker into the lanes at both ends.) 3) Apply fixed voltage of 50 to 150 V and perform electrophoresis. 6.Staining of Bands * When the gel has been prestained with ethidium bromide, perform only step 3. 1) Prepare 1 μg/ml ethidium bromide solution in sufficient quantity to completely immerse the gel and dispense into the gel staining tray. 2) Place the gel into the tray and allow to leave for 20 to 30 minutes. 3) Set the gel on the ultraviolet transilluminator and photograph the gel. Compare the sample DNA bands with DNA marker to confirm the presence and size of DNA bands. Precautions Always use gloves when working with ethidium bromide or handling stained gel. URL:http://www.takara-bio.com 9 Bacteria Screening PCR Kit Cat. #RR114A v201310Da 7.Assessment A band of approximately 419 - 425 bp ( 424 bp in E. coli ) can be detected with ENT primer in the sample containing Enterobacteriaceae family, including E. coli and Salmonella. A band of approximately 380 - 382 bp (381 bp in Bacillus cereus ) can be detected with BS primer when the sample contains genus Bacillus, including Bacillus cereus , or genus Staphylococcus, including Staphylococcus aureus . (For lists of bacteria detected using the ENT and BS primers, refer to Table 1 at VIII. Application Examples.) A band of 424 bp can be detected by the ENT primer when using the Positive Control ENT. A band of 381 bp can be detected by the BS primer when using the Positive Control BS. Electrophoresis results Approx. 420 bp band detected by ENT Primer Approx. 380 bp band detected by BS Primer Assessment Sample contains Enterobacteriaceae family strains, such as E. coli or Salmonella bacteria. Sample contains genus Bacillus strains, such as Bacillus cereus , or genus Staphylococcus strains, such as Staphylococcus aureus . ENT Primer failed to detect any bands Enterobacteriaceae family bacteria in the sample are below the detection limit when 424-bp band is detected in Positive Control ENT. If a 424-bp band is present in the Positive Control ENT, something may be wrong with the PCR detection. Repeat PCR. BS Primer failed to detect any bands Bacteria in Bacillus or Staphylococcus genera in sample are below the detectable range when 381-bp band is detected in Positive Control BS*. If a 381-bp band is not present in the Positive Control BS, something may be wrong with the PCR detection. Repeat PCR. No band detected in Negative Control Indicates no contamination. 420-bp or 380-bp band detected in Indicates possibility of contamination. Perform deconNegative Control tamination of reaction solution mixing area and equipment used. Repeat PCR. *:To improve sensitivity, use the techniques described in Options 1 and 2 of the section "VII-1. Preparation of Sample Solutions" as necessary. 10 URL:http://www.takara-bio.com Cat. #RR114A Bacteria Screening PCR Kit v201310Da VIII.Application Examples Specificity of Primers ENT and BS to various bacteria [ Methods ] PCR was performed with both the ENT and BS primers using 50 pg of genomic DNA (equivalent to approximately 104 copies) as a template. PCR Reaction: 95℃ 60 sec. ↓ 95℃ 30 sec. 59℃ 30 sec. 72℃ 30 sec. ↓ 72℃ 60 sec. 4℃ 30 cycles Agarose electrophoresis analysis was performed on 2 μl of reaction solution after PCR reaction. [ Result ] Figure 1 shows results of agarose gel electrophoresis. Table 1 shows the reaction specificity shown as positive or negative ( + , - ). (Primer Mix ENT was used for reactions involving Enterobacteriaceae (excluding Proteus mirabilis ) and Vibrionaceae family bacteria. Primer BS was used for reactions involving genus Bacillus , Staphylococcus and Aerococcus bacteria.) Fig. 1 PCR Reaction Results for ENT and BS Primers (Electrophoresis) Lane 1:ϕX174 Hae III digest Marker Lane 14:Campylobacter coli DNA Lane 2:Negative Control (No Template DNA) Lane 15:Flavobacterium johnsoniae DNA Lane 3:Serratia ficaria DNA Lane 16:Streptococcus mutans DNA Lane 4:Klebsiella pneumoniae DNA Lane 17:Staphylococcus epidermides DNA Lane 5:Salmonella enteritidis DNA Lane 18:Staphylococcus aureus DNA Lane 6:Escherichia coli DNA Lane 19:Bacillus subtilis DNA Lane 7:Citrobacter freundii DNA Lane 20:Bacillus megaterium DNA Lane 8:Yersinia enterocolitica DNA Lane 21:Bacillus cereus DNA Lane 9:Vibrio vulnificus DNA Lane 22:Aerococcus viridans DNA Lane 10:Photobacterium leiognathi DNA Lane 23:Enterococcus faecalis DNA Lane 11:Aeromonas hydrophila DNA Lane 24:Lactobacillus casei DNA Lane 12:Pseudomonas aeruginosa DNA Lane 25:ϕX174 Hae III digest Marker Lane 13:Alcaligenes faecalis DNA URL:http://www.takara-bio.com 11 Bacteria Screening PCR Kit 1 2 3 4 5 6 Cat. #RR114A v201310Da 7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25 Figure 1-1. ENT Primer Results 1 2 3 4 5 6 7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25 Figure 1-2. BS Primer Results 12 URL:http://www.takara-bio.com Bacteria Screening PCR Kit Cat. #RR114A v201310Da Table 1.Assayed Bacterial Species and Reaction Specificity Family Rhizobiaceae Alcaligenaceae Neisseriaceae Xanthomonadaceae Legionellaceae Pseudomonadaceae Moraxellaceae Vibrionaceae Enterobacteriaceae URL:http://www.takara-bio.com Genus, Species Agrobacterium radiobacte Alcaligenes faecalis Chromobacterium violaceum Neisseria meningitides Xanthomonas maltophilia Legionella pneumophila Pseudomonas aeruginosa Pseudomonas fluorescens Moraxella antipestifer Acinetobacter baumannii Vibrio vulnificus Vibrio parahaemolyticus Photobacterium leiognathi Aeromonas hydrophila (1) Enterobacter agglomerans Enterobacter cloacae Citrobacter freundii Escherichia coli Escherichia coli JM109 Escherichia coli HB101 Escherichia coli 0157:H7 Hafnia alvei Klebsiella pneumoniae Plesiomonas shigelloides Proteus mirabilis Salmonella typhimurium Source of strains ENT* ATCC 19358T - JCM 1474T - JCM 1249T - ATCC 13077T - JCM 1975T - JCM 7571T - ATCC 27843T - JCM 5963T - JCM 9532T - JCM 6841T - JCM 3725T + ATCC 17802T + ATCC 25521T + ATCC 7966T + JCM 1236T + JCM 1232T + JCM 1657T + JCM 1649T + (2) + (2) + (3) + JCM 1666T + JCM 1662T + ATCC 14029T + JCM 1669T - IFo 13245 + BS* - - - - - - - - - - - - - - - - - - - - - - - - - - 13 Bacteria Screening PCR Kit Cat. #RR114A v201310Da (Continued) Family Pasteurellaceae Campylobacteraceae Helicobacteraceae Clostridiaceae Eubacteriaceae Acidaminococcaceae Bacillaceae Listeriaceae Staphylococcaceae Lactobacillaceae Aerococcaceae Enterococcaceae Streptococcaceae Coriobacteriaceae Actinomycetaceae Micrococcaceae Microbacteriaceae Corynebacteriaceae Propionibacteriaceae Bifidobacteriaceae Bacteroidaceae Porphyromonadaceae Prevotellaceae Flavobacteriaceae Flexibacteraceae Fusobacteriaceae 14 Genus, Species Salmonella typhimurium Salmonella enteritidis Serratia ficaria Serratia marcescens Shigella flexneri Yersinia enterocolitica Haemophilus influenzae Campylobacter coli Helicobacter pylori Clostridium perfringens Eubacterium alactolyticum (4) Veillonella alcalescens Bacillus cereus Bacillus cereus Bacillus thuringiensis Bacillus licheniformis Bacillus megaterium Bacillus pumilus Bacillus subtilis Listeria grayi Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermides Lactobacillus casei Lactobacillus gasseri Lactobacillus bulgaricus Aerococcus sp. Aerococcus viridans Enterococcus faecalis Streptococcus mutans Streptococcus salivarius Streptococcus agalactiae Collinsella aerofaciens Actinomyces naeslundii Micrococcus luteus Microbacterium lacticum Corynebacterium kutscheri Corynebacterium xerosis Propionibacterium acnes Bifidobacterium longum Bifidobacterium adolescentis Bacteroides vulgatus Porphyromonas gingivalis Prevotella intermedia Flavobacterium johnsoniae Cytophaga arvensicola Fusobacterium nucleatum Source of strains ENT* BS* IFo 14211 + - IFo 3313 + - JCM 1241T + - JCM 1239T + - ATCC 29903T + - ATCC 9610T + - ATCC 33391T - + ATCC 33559T - + ATCC 43504T - + JCM 1290T - + JCM 6480T - + ATCC 27215 - + IFo 15305T - + ATCC 11950 - + ATCC 10792T - + JCM 2505T - + JCM 2506T - +/- JCM 2508T - + IFo 13719T - + ATCC 19120T - +/- IFo 3060 - + JCM 2874 - + JCM 2414T - + JCM 1134T - - JCM 1131T - - JCM 1002T - - MRA1 (5) - + IFo 12219T - + MRA2 (5) - - JCM 5705T - - JCM 5707T - - JCM 5671T - - JCM 10188T - - JCM 8349T - - JCM 1464T - - JCM 1379 - - JCM 9385T - - JCM 1971T - - JCM 6425T - - JCM 1217T - - JCM 1275T - - JCM 5826T - - ATCC 33277T - - ATCC 25611T - - JCM 8514T - - JCM 2836T - - JCM 2836T - - URL:http://www.takara-bio.com Bacteria Screening PCR Kit Cat. #RR114A v201310Da * : Assessment +: PCR amplified product detected. + / -: Minute quantities of PCR amplified product detected. -: PCR amplified product not detected. Note: (1) While the genus Aeromonas currently belongs to the Vibrionaceae family, there has been discussion of reclassifying it as a member of the Aeromonadaceae family. (Refer to TAXONOMIC OUTLINE OF THE PROCARYOTE GENERA BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, SECOND EDITION Release 1.0, April 2001). (2) Commercially Available Strains for Research. (3) DNA purchased from Nissui Co., Ltd. (Tokyo, Japan) , which is a mixture of ATCC 43889 and ATCC 43890. (4) Reclassified as Pseudoramibacter alactolyticym . Clin Infect Dis 1997 Sep;25 Suppl 2:S78-87 (5) MRA1and MRA2 are isolated and identified by Nisshin Foods. IX.References 1) Rupf, S., Merte, K. and Eschrich, K. (1999) J. Dent. Res . 78: 850-856. 2) Wang, R.-F., Cao, W.-W. and Cerriglia, C.E. (1997) J. Appl. Microbiol . 83: 727-736. X.Related Products O-157 (Verocytotoxin Genes) PCR Screening Set (Cat. #RR100)* *Not available in all geographic locations. Check for availability in your region. URL:http://www.takara-bio.com 15 Bacteria Screening PCR Kit Cat. #RR114A v201310Da NOTICE TO PURCHASER: LIMITED LICENSE [A1] PCR Notice This product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711 and 6,127,155. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Environmental Testing, Food Testing, Industrial Microbiology, including reporting results of purchaser's activities for a fee or other commercial consideration, and also for the purchaser's own internal research. No right under any other patent claim (such as the dsDNAbinding dye process claims in US Patents Nos 5,994,056 and 6,171,785) is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [L33] Bacteria Screening This product is covered by the claims of U.S. Patent No. 7,326,779 and its foreign counterpart patent claims. [M57] LA Technology This product is covered by the claims 6-16 outside the U.S. corresponding to the expired U.S. Patent No. 5,436,149. NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at +81 77 543 7247 or from our website at www.takara-bio.com. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. 16 URL:http://www.takara-bio.com