PCR DIG Probe Synthesis Kit

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1 For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. PCR DIG Probe Synthesis Kit For generation of highly-sensitive probes labeled with DIG-dUTP (alkali-labile) in polymerase chain reaction Cat. No Version Dec Store at 15 to 25 C 1. What this Product Does Number of Reactions The kit is designed for approx. 25 PCR reactions with a final reaction volume of 50 µl each. Contents Vial/ Label Cap 1 Enzyme mix, Expand High Fidelity Contents / Function 30 l enzyme mix (105 U) 3.5 U/l Storage buffer: 20 mm Tris-HCl, ph 7.5 (25 C), 100 mm KCl, 1 mm dithiothreitol (DDT), 0.1 mm EDTA, 0.5% Tween 20 (v/v)*, 0.5% Nonidet P40 (v/v)*, 50% glycerol (v/v). Enzyme mix for the labeling of PCR products 2 PCR DIG probe 125 l synthesis mix, Mixture containing datp, dctp, dgtp 10 conc. (2 mm each); 1.3 mm dttp; 0.7 mm DIG-11-dUTP, alkali-labile; ph 7.0. Nucleotide mix for the labeling of PCR products 3 PCR buffer with 1 ml MgCl 2, Expand High Fidelity buffer 10 conc. 10 conc with 15 mm MgCl 2 Buffer for the PCR labeling reaction 4 dntp stock solution, 10 conc. 5 Control template 6 Control PCR primer mix 125 l Mixture contains datp, dctp, dgtp, dttp (2 mm each), ph 7.0 Solution for the possible dilution of the PCR labeling reaction 50 l 1 ng plasmid DNA [20 pg/l] in Tris/EDTA buffer; ph 8.0 The 5 kb plasmid contains the cdna for human tissue type plasminogen activator (tpa). Template for the control reaction 25 l [50 pmol] of control PCR primer 1 and 2 (2 M each). Primer for the control reaction N Vial 2 (PCR DIG probe synthesis mix) contains a mixture of nucleotides, including the DIG-dUTP. The concentration of DIG-dUTP provided here differs from a separate yet similar reagent, the PCR DIG labeling mix*. These nucleotide mixes should not be interchanged. Vial 2 (PCR DIG probe synthesis mix) contains a higher concentration of DIG-dUTP to achieve maximal DIG incorporation, and maximal probe sensitivity. Only vial 2 should be used in combination with the PCR DIG Probe Synthesis Kit. Storage and Stability The unopened kit is stable at 15 to 25 C through the control date printed on the label. L The kit is shipped on dry ice. Additional Equipment and Reagents Required Template DNA, gene-specific PCR primer pair Water, PCR Grade* Thermal block cycler (e.g., Applied Biosystems GeneAmp PCR System 9600) 0.2 ml thin-walled PCR tubes* Sterile reaction tubes for preparing master mixes and dilutions *available from Application The kit is especially designed for generation of highly sensitive hybridization probes suitable for detection of low (single) copy target sequences. Probes generated with this kit are alkali-labile and can easily be removed from blots (e.g., genomic) prior to rehybridization. 2. How To Use this Product 2.1 Before You Begin PCR Amplification Parameters Optimize PCR amplification parameters (cycling conditions, template concentration, primer sequence, and primer concentration) for each template and primer set in the absence of DIG-dUTP before attempting incorporation of DIG. DIG-dUTP Concentration According to the length of the probe being labeled the concentration of DIG-dUTP has to be adapted: < 1 kb, use a 1:3 ratio of DIG-dUTP : dttp (standard labeling mix vial 2). N DNA containing a high GC content may require a 1 : 6 ratio of DIG-dUTP : dttp. 1 kb, use a 1 : 6 ratio of DIG-dUTP : dttp. Mix equal parts of PCR DIG probe synthesis mix (vial 2) and dntp stock solution (vial 4) 3 kb, use a 1 : 6 ratio of DIG-dUTP : dttp and substitute the Expand Long Template enzyme mix for the Expand High Fidelity enzyme mix included in the PCR DIG Probe Synthesis Kit. N You may possibly need to reduce the DIG-dUTP : dttp ratio as low as 1:10. Sample Material Purified DNA containing the sequence to be labeled. Use either: Plasmid DNA, pg (optimal amount, 10 pg) Genomic DNA, 1 50 ng (optimal amount, 10 ng) L Purity of template is not as critical for PCR labeling as for other types of labeling

2 Unlabeled Positive Control Identical to experimental sample except the reaction mix contains no DIG-dUTP. L Always include this control reaction in every experiment. It is required for evaluating probe labeling efficiency. Labeled Positive Control Substitutes the tissue plasminogen activator (tpa) template and primers (included in the PCR Probe Synthesis Kit) for your experimental template and primers. Produces a labeled probe that recognizes human tpa sequences. The tpa probe generated in the control PCR recognizes a restriction length polymorphism (RFLP) for an Eco RI site in human DNA (5). This results in the detection of variable fragment patterns depending on the human DNA used. One of the following fragment patterns of human single-copy tpa gene should be detected: 2.9 kb kb 2.5 kb kb 2.9 kb kb kb L As you gain experience with the labeling kit, you may choose not to run this control reaction. 2.2 Preparation of Reaction Mixes Thaw the reagents and store on ice. Briefly vortex and centrifuge all reagents before setting up the reactions. Prepare a 10 conc. solution of each respective PCR primer. L If you are using e.g., a final concentration of 0.5 M for each primer, the 10 conc. solution would contain a 5 M concentration of the respective primer. Add the following reagents in a sterile 1.5 ml reaction tube on ice, in the following order: Component DIG labeled probe Unlabeled DNA control Labeled kit control Final conc. Water, PCR Grade variable variable l - PCR buffer with MgCl 2 10 conc. (vial 3) 5 l 5 l 5 l 1 PCR DIG Labeling 5 l - 5 l 200 M Mix (vial 2) dntp dntp stock solution (vial4) Upstream and dowstream primer - 5 l M dntp 5 l 5 l 5 l 5 l 5 l (vial 6) M each Enzyme mix 0.75 l 0.75 l U (vial 1) template DNA variable variable 5 l (vial 5) 1-50 ng gdna pg cdna Final volume 50 l 50 l 50 l 2.3 PCR Gently vortex the mixture to produce a homogeneous reaction, then centrifuge briefly to collect the solution at the bottom of the tube. N Start thermal cycling immediately. Do not store complete reaction mixes on ice. Place your sample in a thermal block cycler and perform PCR. L Cycling conditions depend on the combination of template, primers, and thermal cycler. The conditions given below may not be optimal for your template/primer combination, but are a good starting point for initial experiments. Cycles Time Temp Initial Denaturation 1 2 min 95 C Denaturation Annealing Elongation Denaturation Annealing Elongation s 30 s 2 min s 30 s 2 min + additional 20 s for each successive cycle 2.4 Analysis of Labeled Probe The best way to check the success of the reaction is to run a portion (5 l) of each reaction on an agarose mini gel and then stain the gel with ethidium bromide (EtBr). The tpa control probe will have an apparent size of bp. L The actual size of the amplicon is 442 bp. The presence of DIG in DNA makes it run slower in the gel than unlabeled DNA. The agarose concentration of the gel determines the extent to which the migration rate deviates from its true value. Your unlabeled control probe should be the predicted size. Both the labeled experimental probe and the unlabeled control probe should be clearly visible on the gel. Your labeled experimental probe should migrate slower than your unlabeled control probe (due to the presence of DIG). The EtBr staining of the labeled DNA will be somewhat less than the unlabeled control DNA. L When you are using a high ratio of DIG-dUTP:dTTP (1:3), the reaction will make less labeled probe than unlabeled probe. The polymerase is slowed by the presence of the DIG hapten. 2.5 DNA Electrophoresis, Transfer, and Fixation 95 C 60C 72 C 95 C 60C 72 C Final Elongation 1 7 min 72 C Cooling indefinitely 4 C L The increased elongation time is only required for long (3 kb) fragments. For amplificatin of shorter fragments, use 40 s elongation time for all 30 cycles. The PCR labeled probe should be stored: short term at 2-8 C until the PCR product is used for hybridization. long term at -15 to -25 C, stable for at least one year. General Standard protocols for gel electrophoresis, denaturation and neutralization of the gel are described in the DIG Applications Manual for Filter Hybridization. Gels lacking ethidium bromide are preferred, because ethidium can cause uneven background problems. All common types of DNA transfer methods are suitable for subsequent DIG hybridization (7,8). In our experience, best results are obtained when gels are blotted by capillary transfer with 20 SSC on nylon membranes, positively charged*. N Alkali transfer (e.g., in 0.4 M NaOH) is not suitable for the transfer of DIG-labeled Molecular Weight Markers*. 2

3 Overview Gel electrophoresis. Denaturation and neutralization of DNA in the gel. Fixation of DNA on the membrane. Electrophoresis We recommend the following: Prepare a suitable electrophoresis gel as thin as possible. Load small amounts of the target DNA samples: genomic DNA 1 5 g per lane plasmid DNA 1 ng per lane DIG-labeled Molecular Weight 2 5 l per lane Marker* N Make sure to load enough marker to produce prominent bands that are about the same size as your hybrid. Run the gel until the DNA bands are well separated. To assess the quality of the target DNA, stain the gel briefly in g/ml ethidium bromide, then destain with water. Examine the gel under UV light. Fixation procedure After the transfer, while the blot is still damp, fix the DNA to the blot by either of the following methods: IF you want to... UV-crosslinking (nylon membrane) bake at 120 C (nylon membrane) THEN... Storage of membrane (optional) Please refer to the following table. place the membrane on Whatman 3MM-paper soaked with 2 SSC. UV-crosslink the wet membrane without prior washing. after the UV-crosslinking, rinse the membrane briefly in double distilled water and allow to airdry. wash the membrane briefly in 2 SSC. bake the nylon membrane at 120 C for 30 min or according to the manufacturer`s instructions. IF... THEN... you want to go ahead. use the membrane immediately for prehybridization. you want to work later on store the membrane dry at 2 to 8 C. Hybridization with the DIG-labeled Probe The procedures for using the DIG-labeled probes for the detection of human genomic DNA on a Southern blot can also be used for the detection on Dot blots. We recommend to use DIG Easy Hyb buffer* and Hybridization bags* for best results. For detailed information about the Hybridization protocol please see the DIG Easy Hyb buffer package insert. Overview Do not allow the membrane to dry at any time from the beginning of prehybridization through probe-hybrid visualization. If the membrane dries or sticks to a second membrane (e.g., during simultaneous processing of blots), the assay will have a high background. Prehybridization of blot. Hybridization with DIG-labeled probe. Stringency washes. Storage of Membrane After finishing the last high stringency wash, you may air dry the membranes and store it in a sealed bag at +2 to +8 C for later detection. Chemiluminescent Detection For the chemiluminescent detection of the labeled DIG-probe we recommend to use Anti-digoxigenin-AP, Fab fragments * CDP-Star *or CSPD* DIG Block and Wash Buffer Set* Overview This table lists the single steps of the chemiluminescent detection. L For detailed information about the detection protocol please see the package insert of CDP-Star or CSPD. Washing and blocking of membrane. Antibody binding. Washing and equilibration of membrane. Chemiluminescent reaction. Exposure to imaging instrument or film. Rinse membrane briefly in double distilled water. Stripping and Reprobing of DNA Blots (optional) General The alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization experiment. Additional Reagents Required 0.2 N NaOH, 0.1% SDS (w/v) 2 SSC Protocol Please refer to the following table. N When stripping and rehybridization of blots is planned, the membrane should not dry off at any time. Wash for 2 15 min in 0.2 N NaOH, SDS, 0.1% (w/v) at 37 C under constant agitation. Equilibrate briefly in 2 SSC. Prehybridize and hybridize with the next probe according to the protocol. Probe Concentration The standard probe concentration is 2 l probe per ml hybridization buffer. If the labeled PCR product band on the evaluation gel was very faint, use up to 4 l probe per ml hybridization buffer. If the labeled band was very strong, use only 1 l (or even as little as 0.5 l) probe per ml hybridization buffer. 3

4 3. Troubleshooting Possible Cause Low yield of PCR is not DIG-labeled optimized PCR product Cloudy hybridization background Hybridization smear Too much DIGdUTP in reaction 1. Probe concentration too high in the hybridization solution. Template concentration too high during PCR. Target concentration too high on the blot. Recommendation Always optimize the PCR parameters (cycling conditions, template concentration, primer sequence, and primer concentration) for each template and primer set in the absence of DIG-dUTP before attempting incorporation of DIG. Reduce the concentration of DIG-dUTP in the reaction. This is especially important for long templates, for further information see section 2.1. Reduce probe concentration to 1 l probe per ml DIG Easy Hyb buffer. N Evaluate the amount of labeled probe in the PCR product. If the amount of labeled PCR product band is very strong on the gel, use as little as 0.5 ml probe per ml hybridization buffer. For best results, use only small amounts of template. Ideal amounts: 10 pg plasmid DNA or 10 ng genomic DNA. N Our experience indicates that higher concentrations of template lead to large amounts of primary extension products in the labeled probe. We recommend to use 1 5 g per lane genomic DNA or 1 ng plasmid DNA per lane. 1 DIG-labeled nuceotides in the template slow the polymerase and eventually reduce the ability of the polymerase to synthesize full-length products that contain the primer sequences needed for the start of the next round of amplification. As the template gets longer, the effect increases. 4. Additional Information on this Product Product Description The PCR DIG Probe Synthesis Kit contains all reagents required for the direct digoxigenin (DIG)-labeling of DNA fragments generated by the polymerase chain reaction (PCR) process (1, 2). The polymerase chain reaction is ideally suited to prepare specific and efficiently labeled hybridization probes. The kit enables the synthesis of highly sensitive probes by incorporation of DIG-dUTP into the PCR product. The nucleotide concentration in the PCR DIG Probe Synthesis Mix ensures the detection of single-copy genes in genomic blots after hybridization to DIG-labeled PCR products. The Expand High Fidelity enzyme mix included in this kit ensures maximal yield and highest accuracy. Furthermore a dntp stock solution is supplied to allow adjustment of the concentration of labeled nucleotide. A plasmid containing a cdna for a human single-copy gene and the respective primers serve as a control for the efficiency of the PCR labeling reaction. Synthesis Principle PCR products can directly be amplified and labeled from low amounts of plasmid or genomic DNA and subsequently be used as hybridization probes without further purification. PCR labeling DIG-labeled DNA probes are generated according to the PCR labeling technique. Hybridization DIG-labeled probes are used for hybridization to membrane blotted nucleic acids according to standard methods. The use of the alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization with a second DIGlabeled probe. Immunological detection The hybridized probes are immunodetected with anti-digoxigenin-ap, Fab fragments and are then visualized with a chemiluminescent substrate (CDP-Star* or CSPD*) or by colorimetric detection with e.g., NBT/ BCIP. Labeling Efficiency Optimal reaction conditions are dependent on template DNA and primer. In particular incubation times and temperatures, concentration of Mg 2+ and enzyme but also concentration of template DNA and primer should be optimized for optimal results for each new primer/ template pair (3). Sensitivity The nucleotide concentration in the PCR DIG probe synthesis mix (vial 2) ensures the identification of single-copy genes in genomic blots after hybridization to DIG-labeled PCR products. Human single-copy genes typically are detectable in 5 mg of genomic DNA. References 1 Saiki, R. et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, Lion, T. & Haas, O.A. (1990) Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction. Anal. Biochem. 188, Rolfs, A. et al. (1992) PCR: Clinical Diagnostic and Research, Springer Verlag, Berlin. 4 Innis, M. A. et al. (1990) PCR Protocols, Academic Press Inc., San Diego/CA. 5 Benham, F. J. et al. (1984) Assignment of tissue-type plasminogen activator to chromosome 8 in man and identification of a common restriction length polymorphism within the gene. Mol. Biol. Med. 2, Sambrook, J., Fritsch, E.M. and Maniatis,T. (1989) Molecular cloning: a laboratory manual, 2nd edition,cold Spring Harbor Laboratory, Cold Spring Harbor Labor, New York. 7 Southern E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, Khandijan, E.W. (1987) Optimized hybridization of DNA blotted and fixed to nitrocellulose and nylon membranes. Bio/Technology 5, Yanze, N. et. al. (2001) Conservation of Hox/ParaHox-Related Genes in the Early Development of a Cnidarian. Developmental Biology 236, Ulbrecht, M.et. al. (2000) Cutting Edge: The Human Cytomegalovirus UL40 Gene Product Contains a Ligand for HLA-E and Prevents NK Cell-Mediated Lysis. J. Immunol. 164,

5 Quality Control Function test: Each lot of the PCR DIG Probe Synthesis Kit is function-tested in PCR. Amplification products are assayed in genomic Southern blots. Under PCR conditions described in the this packinsert, the control reaction generates an amplification product of 442 bp. Due to multiple incorporations of DIG-dUTP during the PCR process, the molecular weight of the PCR products is significantly increased compared to the unlabeled PCR product. A specific fragment pattern is detected after hybridization of the PCR product to 10 µg human genomic DNA and chemiluminescent detection as described in this pack insert. 5. Supplementary Information 5.1 Text Conventions To make information consistent and memorable, the following text conventions are used in this package insert: Text Convention Numbered stages labeled,, etc. Numbered instructions labeled,, etc. Asterisk * Use Stages in a process that usually occur in the order listed Steps in a procedure that must be performed in the order listed Denotes a product available from Roche Applied Science Symbols In this package in the following symbols are used to highlight important information: Symbol L N Description Information Note: Additional information about the current topic or procedure. Important Note: Information critical to the success of the procedure or use of the product. 5.2 Ordering Information offers a large selection of enzymes, reagents, and systems for nonradioactive nucleic acid labeling and detection. For a complete overview of our products and for more detailed information please visit and bookmark our DIG Special Interest Site at Kits Single Reagents Available Printed Material Product Pack Size Cat. No. DNA Isolation Kit for Cells and Tissue High Pure Plasmid Isolation Kit 10 isolations for 400 mg tissue or cells 50 purifications 250 purifications High Pure PCR Product 1 kit (50 purifications) Purification Kit 1 kit (250 purifications) High Pure PCR Template Preparation Kit 1 kit (100 reactions) Anti-Digoxigenin-AP, Fab fragments Agarose MP 150 U (200 µl) g 500 g Blocking Reagent 50 g CDP-Star 1 ml 2 ml CDP-Star, ready-to-use 2 50 ml CSPD 1 ml 2 ml 4 ml CSPD, ready-to-use 2 50 ml NBT/BCIP 20 tablets ready-to-use tablets NBT/BCIP Stock solution 8 ml DIG Easy Hyb (readyto-use hybridization solution without formamide) 500 ml DIG Easy Hyb Granules 1 set (6 100 ml) DIG Luminescent Detection Kit 1 kit (50 blots) DIG Wash and Block Buffer Set DNA MWM II, DNA MWM III, DNA MWM V, DNA MWM VI, DNA MWM VII, DNA MWM VIII, 30 blots (10 10 cm2) g (500 l) g (500 l) g (500 l) g (500 l) g (500 l) g (500 l) Hybridization bags 50 bags Nylon Membrane, positively charged 10 sheets,(20 30 cm) sheets, (10 15 cm) roll, (0.3 3 m roll) DIG Product Selection Guide DIG Application Manual for Filter Hybridization Nonradioactive In Situ Hybridization Application Manual 5

6 Disclaimer of License The labeling of nucleic acids with DIG is covered by EP patents and as well as the following US patents , and owned by Roche Diagnostics GmbH. NOTICE TO PURCHASER: LIMITED LICENSE A license under the non-u.s. counterparts of U.S. Patents Nos. 4,683,202, 4,683,195 and 4,965,188 owned by F. Hoffmann-La Roche Ltd. (Roche), for use in research and development, has an up-front fee component and a running-royalty component. The purchase price of the product includes a limited, non-transferable license under the running-royalty component to use only this amount of product to practice the Polymerase Chain Reaction (PCR) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an authorized thermal cycler. This product is also an authorized reagent and may also be used under service sublicenses from Applied Biosystems under the foregoing patents. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted expressly, by implication or estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Contact and Support To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at: To call, write, fax, or us, visit the home page, and select your home country. Countryspecific contact information will be displayed. Use the Product Search function to find Pack Inserts and Material Safety Data Sheets. Roche Diagnostics GmbH Mannheim Germany

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