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RT-PCR Master Mix (2X) Product Number 78370 100 reactions USB, the logo design and ExoSAP-IT are registered trademarks of USB Corporation. Tested User Friendly and FideliTaq are trademarks of USB Corporation. ExoSAP-IT is covered by US Patent Nos. 6,379,940 and 6,387,634. Taq DNA Polymerase—sold under licensing arrangements with Roche Molecular Systems, Inc. Purchase is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F. Hoffmann-La Roche Ltd. FideliTaq™ DNA Polymerase technology is licensed under U.S. Patent 5,436,149 owned by TaKaRa Bio Inc. TRIzol is a registered trademark of Molecular Research Center, Inc. ©USB Corporation 2004—All rights reserved Printed in the United States USB Corporation 26111 Miles Road Cleveland, Ohio 44128 USA www.usbweb.com P-78370A rev 05/04 STORAGE Store at -15°C to -30°C. Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. CONTENTS COMPONENTS Components ....................................................................................................3 All reagents have been extensively tested and carefully prepared to meet USB® standards. USB recommends the reagents be used as directed in order to achieve best possible results. Quality Control ................................................................................................3 Safety Warnings and Precautions ..................................................................3 Description ......................................................................................................4 Materials Not Supplied ...................................................................................6 Protocol ...........................................................................................................7 Supplementary Information ............................................................................8 RNA Quality .................................................................................................8 Amount of RNA per Reaction .......................................................................8 RNase Contamination ..................................................................................9 Primer Design ..............................................................................................9 Thermal Cycler Program Design ................................................................10 Optimizing RT-PCR ....................................................................................10 Suggestions for Difficult Templates .............................................................10 Troubleshooting ............................................................................................11 References ....................................................................................................13 Related Products ..........................................................................................14 The following components are included: RT-PCR Master Mix (2X): A unique, proprietary formulation including M-MLV Reverse Transcriptase, Taq DNA Polymerase, recombinant Ribonuclease Inhibitor, nucleotides, and magnesium in a novel RT-PCR buffer. Magnesium concentration is 3mM in the 2X RT-PCR Master Mix. Product is supplied as 4 x 625 µl tubes, enough for 100 reactions in a 50 µl volume. Magnesium Chloride (25mM), PN 71167 Water, RNase-Free (DEPC-Treated), PN 70783 The enclosed reagents should be stored at -15°C to -30°C (NOT in a frost-free freezer). After thawing for use, keep reagents on ice. QUALITY CONTROL RT-PCR Master Mix (2X) is a Tested User Friendly™ product, assuring reliable results. Release specifications for the RT-PCR Master Mix are based on amplifying a 459 bp β-actin target from 10 pg of human placental total RNA. No contaminating endonucleases, exonucleases, or ribonucleases were detected. Contact Information ......................................................................................17 SAFETY WARNINGS AND PRECAUTIONS Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. USB recommends this product be used only by persons who have been trained in the principles of good laboratory practice. Wear suitable protective clothing such as laboratory coat, safety glasses, and gloves. Avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water (see ‘Material Safety Data Sheet’ for specific advice). 2 3 DESCRIPTION RT-PCR Master Mix (2X) provides maximum convenience and optimal performance for highly sensitive and specific one-step RT-PCR reactions in a single tube. This unique formulation combines all the reagents necessary for successful RT-PCR: M-MLV Reverse Transcriptase, Taq DNA Polymerase, recombinant RNase Inhibitor, Ultrapure nucleotides, and magnesium in a proprietary buffer. Simply add the RT-PCR Master Mix to RNA template, primers, and RNase-free water and the reactions are ready to begin. RT-PCR converts and amplifies single-stranded RNA template yielding doublestranded DNA product. In the RT step, reverse transcriptase synthesizes single-stranded DNA molecules complementary to the RNA template (first-strand cDNA). During the PCR step, a thermostable DNA polymerase first synthesizes second-strand DNA complementary to the first-strand cDNA molecules. This generates a double-stranded DNA template which is exponentially amplified in subsequent rounds of thermal cycling. One-step RT-PCR uses gene specific primers, designed to match RNA/cDNA targets, in a single-tube, one-step reaction. This approach offers tremendous convenience when applied to analysis of single targets from multiple RNA samples. Also, it minimizes the possibility of introducing contaminants into reactions between the RT and PCR steps, since both steps are carried out sequentially without opening reaction tubes between steps(1-5). RT-PCR Master Mix may be used to detect targets whose sizes are generally less than 1.0 kb. For targets greater than 1.0 kb and for high fidelity, use FideliTaq™ RT-PCR Master Mix (PN 71185). Targets are easily detected with the RT-PCR Master Mix, from 10 pg to 1 µg of total RNA or 1 pg to 100 ng of polyA RNA. As shown in Figures 1 and 2, many targets have been detected M NA RT 0 pg pg 00 1 1 1 -R fg Total RNA 1 kb — 500 bp — β-actin (459 bp) 200 bp — from much lower amounts of total RNA, with human β-actin being detected from 100 fg of total RNA which corresponds to about 1/100th the total RNA of a single human cell. The mix is very robust, withstanding repeated freezethaw cycles with no effect on product yield (Fig. 2). USB RT-PCR Master Mix provides reliable performance with minimal optimization. Since the mix is preformulated and thoroughly QC tested, experimental variability is significantly reduced. 1 ng 100 pg 10 pg 1 pg Total RNA M -RNA -RT pre post pre post pre post pre post 1 kb — 500 bp — GAPDH (123 bp) 100 bp — 1 kb — β-actin (459 bp) 500 bp — 200 bp — 10 ng 1 ng 100 pg 10 pg Total RNA M -RNA -RT pre post pre post pre post pre post 1 kb — 500 bp — numb (455 bp) 200 bp — Fig. 2. Stability of the RT-PCR Master Mix with a variety of targets. Three targets were RT-PCR amplified from the indicated amounts of human placental total RNA. RT temperature was 50°C and primers were at 0.8µM. The -RNA control included no RNA in the sample and the -RT control included 100 ng of total RNA but used the Taq PCR Master Mix (PN 71162, without reverse transcriptase) to test for any contaminating genomic DNA in the RNA sample. “Pre” indicates lanes in which the Master Mix was not freeze-thawed and “Post” indicates lanes in which the Master Mix was freeze-thawed 15 times, alternating between dry ice and a room temperature water bath. “M” is the DNA marker lane. Fig. 1. Sensitivity of the RT-PCR Master Mix. A 459 bp fragment of the human β-actin gene was RT-PCR amplified from the indicated amounts of human placental total RNA. RT temperature was 50°C and primers were at 0.8µM. The -RNA control included no RNA in the sample and the -RT control included 100 ng of total RNA but used the Taq PCR Master Mix (PN 71162, without reverse transcriptase) to test for any contaminating genomic DNA in the RNA sample. “M” is the DNA marker lane. Since the typical human cell contains about 10 picograms of total RNA, this target’s detection at 100 femtograms represents about 1/100th of the total RNA of a typical human cell. 4 5 MATERIALS NOT SUPPLIED PROTOCOL Necessary reagents: This standard protocol applies to a single reaction where only template, primers, and water need to be added to the master mix. For multiple reactions, scale-up volume of reaction components proportionally. RNA template: Total RNA and polyA RNA can be prepared by standard methods such as acid-guanidinium thiocyanate-phenol-chloroform extraction(6-7), TRIzol® procedures, anion-exchange resins or columns, or obtained from commercial suppliers. RNA should be highly purified and free of RNase, polysaccharide, and proteoglycan contamination(7). Ideally, RNA should also be free of DNA contamination. Oligonucleotide primers: Oligonucleotide primers can be designed according to standard methods(8). Longer oligonucleotides (i.e., over 25 bases) and those with higher melting temperatures (i.e., above 60°C) are recommended to achieve more specific and robust amplification. Gene specific primers which flank an intron or cross an exon-exon border are useful as a control to distinguish amplification from RNA versus contaminating DNA. Optional reagents: Enhancing additives: Solvents such as dimethyl sulfoxide (DMSO), glycerol, trehalose, and betaine can improve results for RT-PCR on longer targets and those with a high degree of secondary structure(9-12). Necessary equipment: Liquid handling supplies such as PCR-grade thin-walled tubes, pipettes, pipettors, and a microcentrifuge are required. Use plastic tubes and pipette tips, certified RNase-free, in order to prevent RNase contamination of samples. Also, the use of barrier-tip pipettes and dedicated PCR pipettors are strongly recommended in order to avoid RNase contamination. Latex gloves (powder-free) should be used for handling reagents and equipment in order to decrease the probability of introducing RNases into samples. Thermal cycler for incubations between 4°C and 95°C is required. Equipment such as a standard horizontal gel apparatus and a UV transilluminator or fluorescence image scanner can be used for analysis of RT-PCR products. 6 1. Thaw reagents at room temperature. Mix thoroughly and then place on ice immediately after thawing. 2. Assemble reaction tubes on ice whenever possible to avoid premature, nonspecific polymerase activity. 3. The following table shows recommended component volumes: Components Vol. for 25 µl reaction Vol. for 50 µl reaction RT-PCR Master Mix (2X) 12.5 µl 25 µl Final Concentration 1X 10µM Forward Primer 10µM Reverse Primer Template RNA Water, RNase-Free 0.1-1.0µM 0.1-1.0µM as needed NA 0.25-2.5 µl 0.25-2.5 µl ≥ 1 µl up to 25 µl 0.5-5.0 µl 0.5-5.0 µl ≥ 1 µl up to 50 µl NOTE: In general, use greater than 0.5µM primers for sensitivity and less than 0.5µM for specificity. 4. Ensure reactions are mixed thoroughly by pipetting or gentle vortexing followed by a brief spin in a microcentrifuge. 5. Optional Optional-Overlay reactions with one-half volume PCR-grade mineral oil (PN 71600) when not using heated lid on thermal cycler. 6. Transfer tubes on ice into a thermal cycler pre-warmed at the reverse transcription temperature for best results. The following table shows recommended cycling conditions: Cycle Name Reverse Transcription Temperature 42-50°C Time 15-30 minutes Initial Denature Denature Anneal 94-95°C 2-3 minutes 94-95°C 55°C 30 seconds 30 seconds Options Use higher temperatures for difficult targets. Most targets generally require a 15 minute incubation period. Initially, annealing temperature should be 5°C below Tm of primers. Extend 68°C 30-60 Extension time should be about 1 minute per kb of seconds expected product length. Repeat previous three cycles as necessary, generally 30-45 times. Final Extend 68°C 5 minutes Final Soak 4-10°C as necessary 7 7. Analyze sample (typically 1 to 10 µl aliquots) by agarose gel electrophoresis. Visualize products with DNA intercalating dyes and a UV transilluminator or fluorescence imager. NOTE: One can test for the presence of contaminating genomic DNA in the RNA sample by using USB Taq PCR Master Mix, PN 71162/71163. Since there is no reverse transcriptase in this mix, any observed product must have been generated from a DNA source. SUPPLEMENTARY INFORMATION RNA Quality The quality of the RNA sample is the most important factor affecting the outcome of RT-PCR reactions. Quality can be defined in terms of both purity and integrity (i.e., proportion of the RNA sample which is full-length). Many purification methods can be used to prepare RNA, provided the methods yield RNA which is essentially free of contaminating DNA, proteins, polysaccharides or proteoglycans, phenol, ethanol, and salts. These contaminants inhibit the activity of M-MLV Reverse Transcriptase, which reduces RT-PCR amplification yields(7). The purity of the RNA sample should be estimated by measuring its absorbance at 260 and 280 nm in TE buffer. A260/A280 ratios should fall between 1.7-2.1. If ratios fall outside this range, re-precipitation of the RNA may be necessary. The integrity of the RNA may be assessed by performing denaturing agarose gel electrophoresis. High-quality, full-length RNA resolves into two discrete bands, composed of 28S and 18S ribosomal RNA, with minimal smearing below each band. RNA of the highest quality has a 28S rRNA band which stains about twice as intense as the 18S rRNA band. Maintain RNA integrity by storing samples in TE (10mM Tris-HCl (pH 7), 1mM EDTA) or 0.1mM EDTA at -80°C, which reduces metal-catalyzed hydrolysis of RNA. Reduce or eliminate any suspected genomic DNA contamination by treating the RNA sample with USB recombinant DNase I (PN 78311). This is particularly important if experiments generate targets of the same size from RNA and contaminating genomic DNA (i.e., no intron present in genomic DNA target). Amount of RNA per Reaction Total RNA may be used at 1 pg to 1 µg per reaction and polyA RNA may be used at 100 fg to 100 ng per reaction. Greater representation of the specific target within a population of RNA molecules and a shorter length of the designed RT-PCR product allow use of lower amounts of RNA. Since polyA RNA comprises approximately 1 to 5% of total RNA, a specific target will be more abundant in polyA RNA than in total RNA. For best results, when working 8 with dilute stocks of RNA (less than 100 ng/µl), freshly prepare the dilute stocks from concentrated stocks rather than subjecting dilute stocks to multiple freezethaw cycles. RNase Contamination In order to avoid RNase contamination, maintain a clean, dust-free work area, use powder-free latex gloves, dedicate a set of pipettors and a microcentrifuge specifically for RNA work, use certified RNase-free barrier tips and plastic tubes, and store RNA in fairly concentrated forms (~ 1 µg/µl) at -80°C(13). Ideally, use solutions that are certified RNase-free or that have been DEPC-treated to inactivate any RNases present. By avoiding potential sources of RNase contamination (ungloved hands, contaminated pipettors, etc.), it is possible to work with RNA without difficulty or inconvenience. Primer Design During one-step RT-PCR, two gene specific primers are designed per RT-PCR product. The 3' or reverse primer is complementary to the target RNA. The 5' or forward primer is complementary to the corresponding first strand of cDNA. In reverse transcription, the reverse primer initiates synthesis of the first strand of cDNA. In PCR, the forward primer initiates synthesis of the second strand of cDNA during the first PCR cycle. Both primers then exponentially amplify the two DNA strands during subsequent PCR cycles. General rules for designing primers can be found in many texts(8). Briefly, primers should be designed to specifically match the desired target and not other sequences present in the target RNA and cDNA. In general, primers should range in length from 18 to 30 nucleotides, exhibit G+C content similar to each other (and ideally in the range of 40 to 60%), and exhibit Tm values ranging from 55 to 65°C that are closely matched to each other. Tm values may be estimated using the following equation: Tm(°C) = 2(A+T) + 4(G+C). More accurate methods for calculation of Tm values may also be applied(8). Primers that do not fit these criteria may also function well, but empirical testing is required. Use of computer programs designed to select appropriate primers in a given sequence is highly recommended. It is useful to design primer sets that give different amplification products from messenger RNA versus genomic DNA that may be a sample contaminant. Whenever possible, primers should flank an intron or span an exon-exon border. For primers flanking an intron, the PCR product will be smaller from RNA compared to contaminating genomic DNA. For primers crossing an exon-exon border, PCR product should not be generated from genomic DNA. Be aware that common housekeeping genes such as β-actin or GAPDH have intron-less pseudogenes in many organisms. In those cases, it is important to have RNA which is free of contaminating genomic DNA. 9 Thermal Cycler Program Design One-step RT-PCR involves use of a single thermal cycler program for reverse transcription, inactivation of reverse transcriptase, and PCR. The thermal cycler program in the Protocol is a good starting point for program design. Several variations may improve results. For targets with high G+C content or suspected to have strong secondary structure, increase the reverse transcription temperature to as much as 50°C. For primers with relatively high Tm values, increase the annealing temperature to about 65°C or use a combined annealing/extension step of 68°C. For targets longer than about 1 kb, increase the extension time to about 2 minutes per kb. Optimizing RT-PCR Addition of supplemental magnesium chloride may also improve results. Benefits are usually observed within a narrow range and concentrations above 5mM should be avoided. TROUBLESHOOTING Problem Possible causes and solutions No Product or Faint Product 1. Use more RNA template, between 1 ng to 1 µg total RNA or 100 pg to 100 ng polyA RNA. Verify integrity of RNA by gel electrophoresis. Confirm concentration of RNA with spectrophotometer. If RNA has been degraded, prepare fresh stock. Improve the sensitivity and yield of RT-PCR reactions by increasing the primer concentrations to above 0.5µM. There is occasionally some loss of specificity when adding more primers, but this is often overcome by increasing the annealing temperature by increments of approximately 2°C. If specificity is preferred rather than sensitivity, decrease the concentration of primers in the RT-PCR reaction to between 0.1-0.2µM. It may also be necessary to design multiple primer sets for each target, at varying distances along the transcript, to obtain best possible results. Another approach is to raise the RT reaction temperature to 50°C, which melts RNA secondary structure and provides for better first-strand cDNA synthesis. If these suggestions fail, try FideliTaq RT-PCR Master Mix (PN 71185), as this mix occasionally yields less background than the standard RT-PCR Master Mix. 2. Remove possible RT inhibitors such SDS, EDTA, salts, etc. from RNA sample by re-precipitation. Suggestions for Difficult Templates 9. Try using FideliTaq RT-PCR Master Mix (PN 71185). Successful amplification with the RT-PCR Master Mix often requires little or no optimization. If targets have a high-degree of secondary structure and/or high G+C content (e.g., > 60%), adding certain supplements, such as DMSO, glycerol, trehalose, and/or betaine to the RT-PCR reaction may improve results(9-12). DMSO and glycerol may be added at final concentrations ranging from 1 to 10% (v/v)(10). Trehalose may be added to 0.6M final concentration(9). Betaine (5M stock, PN 77507) may be added at 0.5M to 2.0M final concentration(9, 12). Betaine and trehalose have been reported to thermostabilize proteins in general(9, 11). Thus, the RT temperature may be elevated in their presence, possibly melting secondary structure. All of these solvents tend to decrease Tm values for double-stranded DNA, thus its presence in reactions may result in a decrease in the optimum annealing temperature by several degrees. If these suggestions fail, try FideliTaq RT-PCR Master Mix (PN 71185) as this mix occasionally yields more product from longer and/or more G/C-rich targets. 10. If template and/or primers exhibit G+C content greater than ~60%, supplement reactions with additives suggested in Supplementary Information section. In general, the optimal amount of additives for a given primer/template combination needs to be determined empirically. 10 3. Increase number of cycles, up to 45 cycles, for low-copy targets. 4. Increase the concentration of primers, for example, from 0.4µM to 0.8µM. 5. Increase the RT step temperature to 50°C to melt RNA secondary structure. 6. Increase MgCl2 final concentration in 0.25mM increments. 7. Test a range of PCR annealing temperatures. Start with an annealing temperature 5-10°C below primer Tm and increase in 1-2°C increments. Annealing temperatures that are either too high or too low can result in absence of product. 8. For targets between 1-2 kb, use at least one minute per kb. 11. Mix RT-PCR Master Mix and reactions well. Spin down contents to bottom of tube. 12. Design new primers. Use oligo design computer program if at all possible. Nonspecific bands and/or background smearing 1. Use less RNA template. 2. Reduce number of cycles. 3. Raise annealing temperature in 1-2°C increments. 11 4. Decrease the concentration of primers, for example from 0.4µM to 0.1µM. Also, do not use oligo (dT) or random primers in one-step RT-PCR reactions. 5. Check for contaminating genomic DNA by using USB Taq PCR Master Mix, PN 71162/71163. If products of expected size are still generated, pre-treat RNA samples with RNase-free DNase I (PN 78311). Also, design primers which flank an intron or span an exon-exon border. 6. Try using FideliTaq RT-PCR Master Mix (PN 71185). 7. Supplement reactions with additives that improve amplification of G+C rich templates. 8. Check integrity of RNA stocks by gel electrophoresis. Prepare fresh dilutions of RNA stocks. 9. Design new primers. Use oligo design computer program if at all possible. If problems persist please contact USB Technical Support for assistance at (800) 321-9322 or [email protected]. Additional information such as new product listings, updated protocols and TechTips, may be found at our website, www.usbweb.com. For technical support outside the U.S., please visit our website for up-to-date contact information on the USB product distributor within your area. REFERENCES 1. GOBLET, C., PROST, E., AND WHALEN, R. G. (1989) Nucleic Acids Res. 17, 2144. 2. SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 8.46-8.53. 3. SELLNER, L. N., COELEN, R. J., AND MACKENZIE, J. S. (1992) Nucleic Acids Res. 20, 1487-1490. 4. ROTH, M. J., TANESE, N., AND GOFF, S. P. (1985) J. Biol. Chem. 260, 9326-9335. 5. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988) Science 239, 487491. 6. PUISSANT, C. AND HOUDEBINE, L.-M. (1990) BioTechniques 8, 148-149. 7. SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 7.9-7.12. 8. SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 8.13-8.16. 9. CARNINCI, P., NISHIYAMA, Y., WESTOVER, A., ITOH, M., NAGAOKA, S., SASAKI, N., OKAZAKI, Y., MURAMATSU, M., AND HAYASHIZAKI, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 520-524. 10. SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 8.9. 11. SANTORO, M. M., LIU, Y., KHAN, S. M., HOU, L. X., AND BOLEN, D. W. (1992) Biochemistry 31, 5278-5283. 12. SPIESS, A.-N. AND IVELL, R. (2002) Anal. Biochem. 301, 168-174. 13. SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 7.82-7.84. 12 13 RELATED PRODUCTS Ultrapure Nucleotides Reverse Transcription Enzymes and RT-PCR Products Product M-MLV Reverse Transcriptase AMV Reverse Transcriptase Ribonuclease Inhibitor, Recombinant (40 units/µl) Oligo dT(12-18) Primer One-Step RT-PCR Kit Two-Step RT-PCR Kit RT Script Kit DNase I, Recombinant Application cDNA synthesis, low RNaseH activity cDNA synthesis Stabilize RNA Priming synthesis of cDNA from mRNA RT-PCR, analysis of multiple templates RT-PCR, analysis of multiple genes RT synthesis of cDNA for cloning, arrays, and RT-PCR Removal of DNA prior to RT-PCR Pack size 25,000 units 100,000 units 200 units 1,000 units 5,000 units Product number 70456Y 70456Z 70041Y 70041Z 71571 100 µl (2.5 nmol) 50 reactions 77405 78350 50 RT/100 PCR rctns 50 reactions 78355 1,000 units 2,500 units 78311 Pack size 50 units 250 units 1,000 units 5 × 250 units 5,000 units 50 units 250 units 1,000 units 5 × 250 units 5,000 units 100 rctns Product number 71160 100 rctns (125 units) 100 rctns 71162 100 rctns 71183 100 rctns 500 rctns 2,000 rctns 5,000 rctns 78200 78201 78202 78205 78360 PCR Enzymes and Related Products Product Taq DNA Polymerase FideliTaq™ DNA Polymerase Taq PCR Kit Taq PCR Master Mix (2X) FideliTaq PCR Master Mix (2X) FideliTaq PCR Master Mix Plus ExoSAP-IT® 14 Application PCR PCR PCR, including all necessary reagents PCR reaction mix (2X), ready-to-use PCR reaction mix (2X), ready-to-use PCR reaction mix, ready-to-use Clean-up of PCR products Product PCR Nucleotide Mix, 10mM each of dATP, dCTP, dGTP, and dTTP PCR Nucleotide Mix, 25mM each of dATP, dCTP, dGTP, and dTTP dATP, dCTP, dGTP, dTTP (Set of Four), 2'-DeoxyNucleoside-5'-Triphosphates, 100mM Solution Application RT and/or PCR, nucleotides Pack size 500 µl Product number 77212 RT and/or PCR, nucleotides 500 µl 77119 RT and/or PCR, nucleotides 4 dNTPs per pack 4 × 25 µmol (250 µl) 1 pack 77100 Application Supplement for RT and/or PCR Supplement for RT and/or PCR Supplement for RT and/or PCR Supplement for RT and/or PCR Supplement for RT and/or PCR Pack size 1 ml 5 x 1 ml 500 ml 1L 1.5 ml 5 x 1.5 ml 10 gm 100 gm 50 mg 5 x 50 mg Product number 71167 Additives for PCR Product Magnesium Chloride, 25mM Solution Glycerol, Nuclease-Free, Ultrapure Betaine, 5M Solution, Ultrapure Trehalose, Dihydrate BSA, 50 mg/ml Solution, Non-Acetylated, Ultrapure 16374 77507 22515 10921 Ultrapure RNA Reagents 71180 71161 Product Diethyl Pyrocarbonate (DEPC) RNA Solutions Kit TE Buffer (1X) Water, RNase-Free 71182 Water, RNase-Free, DEPC-Treated Application RNase inactivation Storage and handling of RNA Storage of RNA/DNA Pack size 25 ml 100 ml 8 × 100 ml per pk, 1 pk 10 × 1 ml 100 ml 500 ml 10 × 1 ml 100 ml 500 ml 1L 5L 10 × 1 ml 100 ml 500 ml 1L Product number 14710 75903 75893 71783 70783 15 USB CORPORATION Ultrapure Electrophoresis Reagents Product Agarose - Separation ≥ 500bp Application Gel electrophoresis Ethidium Bromide Tablets (100 mg/tablet) TAE Buffer (50X) TBE Buffer (5X) Staining RNA and DNA 16 Gel electrophoresis Gel electrophoresis Pack size 25 gm 100 gm 250 gm 500 gm 10 × 100 mg Product number 75817 100 ml 1L 5L 74015 75891 USA Cleveland, Ohio (800) 321-9322 www.usbweb.com 75809 USB products distributed outside the USA: Please visit the USB website at www.usbweb.com for up-to-date contact information within your area. 17 18 19 TLV ACGIH TLV - TWA: 10mg/m3 (total particulate) OSHA TWA: 15mg/m3 (total dust) CHIP R & S PHRASES R:36/37/38 Irritating to eyes, respiratory system and skin. S:26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S:36/37 Wear suitable protective clothing and gloves. EEC NUMBER None CHIP: Irritant. HCS: Irritant. EYES: Flush with water for 15 minutes. Seek medical advice if irritation persists. SKIN: Flush with water, then wash thoroughly with soap and water. Remove contaminated clothing and wash before reuse. Seek medical attention if irritation persists. INHALATION: Remove the victim from exposure and move to fresh air. If breathing is difficult, give oxygen. If not breathing, give artificial respiration. Keep victim quiet and warm. Seek immediate medical attention. INGESTION: Drink water and seek immediate medical attention. Avoid alcoholic beverages. Never give anything by mouth to an unconscious person. Use media suitable to extinguish the supporting or surrounding fire. Wear NIOSH (or equivalent) approved self contained breathing apparatus. For small fires only: use carbon dioxide, dry powder or foam. Contact with strong oxidizing agents may produce an explosion. Explosion Limits for Glycerol = Lower - 1.1; Upper - Not available. Flash point for Glycerol = 193°C (379.4°F); Autoignition temperature for Glycerol = 400°C (752°F) Wear suitable protective clothing including lab coat, safety glasses and gloves to clean small releases. Ventilate the area and stop the leak if it can be done without risk, dilute with water before mopping or take up with sand, earth, or other absorbing material. Place material in a suitable dry, leak-proof waste container. Avoid contact of material with skin or eyes. Use adequate ventilation. %WT > 1% (Exact % considered trade secret) EMERGENCY CONTACT: Chemtrec (800) 424-9300 Outside USA and Canada (703) 527-3887 PRODUCT CODE 78370 Wash thoroughly after handling. Use with adequate ventilation. Wash clothing before reuse. Store at -20°C. Containers (even empty) may retain product vapors and residue. Store away from ignition sources and excess heat. Store away from incompatible materials including strong oxidizers, mixtures with hydrogen peroxide, potassium permanganate, calcium hypochlorite, nitric acid, sulfuric acid, perchloric acid and lead oxide. PERSONAL PROTECTION Wear appropriate personal protective equipment and clothing including lab coat, safety glasses, gloves and NIOSH-approved respirator. A qualified industrial hygienist should evaluate the need for respiratory protection. Use respiratory protection approved by NIOSH (or equivalent) and appropriate to the hazard. Avoid contact of material with skin or eyes. Mechanical ventilation or local exhaust as needed to control exposure to dust, vapors or mists. Access to a safety shower and eye-wash. PHYSICAL AND CHEMICAL For Glycerol: Boiling Point = 288°C. Solubility = Miscible in water. Melting Point = 20°F. Decomposition PROPERTIES Temp. = 290°C. Vapor Pressure = .0025 mm Hg@ 5. Specific Gravity = 1.26. Vapor Density = 3.17 (H2O = 1). Percent Volatile = No data available. Evaporation Rate = No data available. Formula = C3-H8-O3. Appearance = Clear viscous solution. STABILITY AND REACTIVITY Product is stable under normal conditions. Avoid strong oxidizing agents including mixtures with hydrogen peroxide, potassium permanganate, calcium hypochlorite, nitric acid, sulfuric acid, perchloric acid and lead oxide. Contact with Sodium Hypochlorite and Hypochlorous acid may cause an explosion. TOXICOLOGICAL INFORMATION EFFECTS OF OVEREXPOSURE TO GLYCEROL: EYES: Contact may cause irritation and slight corneal injury. SKIN: Prolonged contact may cause irritation and/or allergic reaction. INHALATION: No known toxicity, but excessive fumes may cause irritation if inhaled. INGESTION: May cause irritation of gastrointestinal tract and diarrhea. ADDITIONAL INFORMATION: May cause slight or transient irritation to eyes and skin. Has caused moderate irritation in dermal (rabbit) studies. Low single and repeated dose toxicity. Ingesting large quantities may cause nausea and vomiting. Irritation, mutation, reproductive effects and toxicity data for Glycerol listed in RTECS under MA8050000. See RTECS for complete information. Toxicity data for Glycerol: Oral Mouse LD50 = 4090 mg/kg; Oral Rat LD50 = 12600 mg/kg. Definition(s): RTECS = Registry of Toxic Effects of Chemical Substances. ECOLOGICAL INFORMATION No information available. DISPOSAL CONSIDERATIONS Dispose of material in accordance with applicable local, state, federal regulations. TRANSPORTATION INFORMATION US DOT / IATA: No information available. REGULATORY INFORMATION RCRA - No applicable information. SARA 302 - This material does not have an RQ or TPQ. SARA 313 - This material is not reportable under 313. EPA TSCA Section 8(b) - For Glycerol: Chemical Inventory. Exposure Limits Glycerol - ACGIH TLV TWA: 10mg/m3 (total particulate). OSHA PEL TWA: 15mg/m3 (total dust). California Proposition 65 - No applicable information. This data sheet is based upon information believed to be reliable. The Company makes no statement or warranty as to the accuracy or completeness of the information contained herein which is offered for your consideration, investigation and verification. Any use of the information contained in this data sheet must be determined by the user to be in accordance with appropriate applicable regulations. HANDLING AND STORAGE ACCIDENTAL RELEASE MEASURES FIRE-FIGHTING INFORMATION HAZARDS IDENTIFICATION FIRST-AID MEASURES Revision: 03/22/2004 Hazard information is provided for compliance with both the UK Chemicals (Hazard Information and Packaging) (CHIP) Regulations and the US Hazard Communication Standard (HCS) IDENTIFICATION OF THE PRODUCT NAME SUBSTANCE/PREPARATION RT-PCR Master Mix (2X) AND COMPANY SUPPLIER: USB Corporation 26111 Miles Road, Cleveland, OH 44128 Phone: (216) 765-5000 COMPOSITION/ HAZARDOUS COMPONENTS HAZARD CAS NO. Glycerol 56-81-5 Material Safety Data Sheet