Download RT-PCR Master Mix (2X)

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
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ExoSAP-IT®
14
Application
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(Set of Four), 2'-DeoxyNucleoside-5'-Triphosphates,
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Application
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Additives for PCR
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Magnesium Chloride,
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Glycerol, Nuclease-Free,
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Betaine, 5M Solution,
Ultrapure
Trehalose, Dihydrate
BSA, 50 mg/ml Solution,
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22515
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15
USB CORPORATION
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16
Gel electrophoresis
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100 ml
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74015
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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