SuperScript ™ III First-Strand Synthesis System for RT-PCR
Cat. No: 18080-051 Size: 50 reactions
Store at -20°C
Description
The SuperScript ™ III First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A)+ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 1 pg to 5 µg of total RNA. SuperScript ™ III Rever Transcripta is a version of M-MLV RT that has been engineered to reduce RNa H activity and provide
incread thermal stability. The enzyme is ud to synthesize cDNA at a temperature range of 42–55°C, providing incread specificity, higher yields of cDNA, and more full-length product than other rever transcriptas. Becau SuperScript ™ III RT is not significantly inhibited by ribosomal and transfer RNA, it may be ud to synthesize first-strand cDNA from a total RNA preparation.
cDNA synthesis is performed in the first step using either total RNA or poly(A)+-lected RNA primed with oligo(dT), random primers, or a gene-specific primer. In the cond step, PCR is performed in a parate tube using primers specific for the gene of interest. For the PCR
reaction, we recommend one of the following DNA polymeras: Platinum ® Taq DNA Polymera provides automatic hot-start conditions for incread specificity up to 4 kb, Platinum ® Taq DNA Polymera High Fidelity provides incread yield and high fidelity for targets up to 15 kb, and Platinum ® Pfx DNA Polymera provides maximum fidelity for targets up to 12 kb. System Component Amount
Oligo(dT)20 (50 µM) 50
µl Random hexamers (50 ng/µl) 250 µl
10X RT buffer* 1 ml
25 mM MgCl 2 500 µl
0.1 M DTT 250 µl
10 mM dNTP mix 250 µl SuperScript ™
III RT (200 U/µl) 50 µl
RNaOUT ™ (40 U/µl) 100 µl
E. coli RNa H (2 U/µl) 50 µl DEPC-treated water 1.2 ml Total HeLa RNA (10 ng/µl) 20 µl Sen Control Primer (10 µM) 25 µl Antin Control Primer (10 µM) 25 µl
*200 mM Tris-HCl (pH 8.4), 500 mM KCl Related products Amount Catalog No.
Platinum ® Taq DNA Polymera 100 units 10966-018 250 units 10966-026 500 units 10966-034 Platinum ®
Taq DNA Polymera High Fidelity 100 units 11304-011 500 units 11304-029 Platinum ® Pfx DNA Polymera 100 units 11708-013
250 units 11708-021 500 units 11708-039 PCR x Enhancer System 250 rxns 11495-017 Micro-to-Midi Total RNA Purification System 50 rxns 12183-018 TRIzol ®
Reagent 100 ml 15596-026
200 ml 15596-018 DNa I, Amplification Grade 100 units 18068-015
Custom Primers to order, visit
Quality Control
A minimum of 25 ng of a 353-bp RT-PCR product was obtained from 100 pg of total HeLa RNA and human β-actin primers. A minimum of 25 ng of a 6.8-kb RT-PCR product was obtained from 500 ng of total HeLa RNA and human Pol ε primers.
Summary of Procedure
Denature:
RNA + Primer + dNTPs (10 m l)
qq防沉迷65o
C
Add 10 m
l cDNA Synthesis Mix
o
50C for 50 min 85o
C
Add 1 m l of RNa H
37o
C Anneal:
cDNA Synthesis:Terminate Reaction:Remove RNA:
Add 10 m Remove 2 m l aliquot for PCR (up to 10 m l may be ud)
PCR Amplification:
cDNA Synthesis Mix:
10X RT Buffer 225 mM MgCl
240.1 M DTT
2RNaOUT Ô
1SuperScript Ô III RT
110 m l
18080051.pps
Rev. date: 3 Oct 2003
Page 2 of 4
Recommendations and Guidelines for First-Strand Synthesis
RNA
• High-quality, intact RNA is esntial for full-length, high-quality
cDNA synthesis. This kit is designed for u with 1 pg to 5 µg of total RNA or 1 pg to 500 ng of poly(A)+ RNA. For >5 µg total RNA, increa reaction volumes and amount of SuperScript ™ III RT proportionally. • RNaOUT ™ Recombinant RNa Inhibitor has been added to the
system to safeguard against degradation of target RNA due to ribonuclea contamination of the RNA preparation. •
To isolate total RNA, we recommend the Micro-to-Midi Total RNA Purification System (Cat. no. 12183-018), TRIzol ® Reagent (Cat. Nos. 15596-026/-018), or the Chomczynski and Sacchi method. Oligo (dT)-lection for poly(A)+ RNA is typically not necessary, although it may improve the yield of specific cDNAs. •
Small amounts of genomic DNA in the RNA preparation may be amplified along with the target cDNA. If your application requires removal of all genomic DNA from your RNA preparation, we recommend using DNa I, Amplification Grade (Catalog no. 18068-015). DNa I, Amplification Grade, has been extensively purified to remove trace ribonuclea activities commonly
associated with other “RNa-free” enzyme preparations, and does not require the addition of placental RNa inhibitor.
RNa H Digestion
The nsitivity of the PCR step can be incread (especially for long
templates) by removing the RNA template from the cDNA:RNA hybrid molecule by digestion with RNa H after first-strand synthesis. Prence of RNa H during first-strand synthesis will degrade the template
mRNA, resulting in decread full-length cDNA synthesis and decread yields of first-strand cDNA. The SuperScript ™ III First-Strand Synthesis System introduces RNa H activity only when it is beneficial, and thus offers a unique procedural advantage over other methods.
Primers
The first-strand cDNA synthesis reaction can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs): •
Random hexamers are the most nonspecific priming method, and are typically ud when the mRNA is difficult to copy in its entirety. With this method, all RNAs in a population are templates for first-strand cDNA synthesis, and PCR primers confer specificity during PCR. To maximize the size of cDNA, you should determine the ratio of random hexamers to RNA empirically for each RNA preparation.
Note: For most RT-PCR applications, 50 ng of random hexamers per 5 µg of total RNA is adequate. Increasing hexamers to 250 ng per 5 µg of RNA may increa yield of small PCR products (<500 bp), but may decrea the yield of longer PCR products and full-length transcripts. •
Oligo(dT), a more specific priming method, is ud to hybridize to 3´ poly(A) tails, which are found in the vast majority of eukaryotic mRNAs. Since poly(A)+ RNA constitutes approximately 1% to 2% of total RNA, the amount and complexity of cDNA is considerably less than with random hexamers. We recommend using oligo(dT)20 (provided in the kit).
Note: Oligo(dT) is recommended over random hexamers or GSPs when performing RT-PCR with new mRNA targets. Oligo(dT) produces an RT-PCR product more consistently than random hexamers or GSPs.
•
The most specific priming method us a gene-specific primer for the quence of interest. First-strand synthesis can be primed with the PCR primer that hybridizes nearest to the 3´ terminus of the mRNA. Note that some GSPs fail to prime cDNA synthesis even though they work in PCR on DNA templates. If gene-specific priming fails in RT-PCR, repeat first-strand synthesis using oligo(dT) as the primer.
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First-Strand cDNA Synthesis
The following procedure is designed to convert 1 pg to 5 µg of total RNA or 1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA: 1. Mix and briefly centrifuge each component before u. 2.
Combine the following in a 0.2- or 0.5-ml tube:
Component Amount up to 5 µg total RNA n µl
Primer* 1 µl
*50 µM oligo(dT)20, or
2 µM gene-specific primer (GSP), or
50 ng/µl random hexamers
10 mM dNTP mix 1 µl DEPC-treated water to 10 µl
3. Incubate at 65°C for 5 min, then place on ice for at least 1 min.
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Prepare the following cDNA Synthesis Mix, adding each component in the indicated order.
Component 1 Rxn 10 Rxns 10X RT buffer 2 µl 20 µl
25 mM MgCl 2 4 µl 40 µl 0.1 M DTT 2 µl 20 µl
RNaOUT ™ (40 U/µl) 1
µl 10 µl SuperScript ™
III RT (200 U/µl) 1 µl 10 µl 5. Add 10 µl of cDNA Synthesis Mix to each RNA/primer mixture, mix gently, and collect by brief centrifugation. Incubate as follows.
Oligo(dT)20 or GSP primed: 50 min at 50°C
Random hexamer primed: 10 min at 25°C, followed by
50 min at 50°C
6. Terminate the reactions at 85°C for 5 min. Chill on ice.
7. Collect the reactions by brief centrifugation. Add 1 µl of RNa H to each tube and incubate for 20 min at 37°C.
8.
cDNA synthesis reaction can be stored at -20°C or ud for PCR immediately.
Amplification of Target cDNA
The first-strand cDNA obtained in the synthesis reaction may be amplified directly using PCR. We recommend using 10% of the first-strand reaction (2 µl) for PCR. However, for some targets, increasing the amount of first-strand reaction up to 10 µl in PCR may result in incread product yield.
We recommend the following DNA polymeras (for ordering information,
e page 1): • Platinum ® Taq DNA Polymera provides automatic hot-start conditions for incread specificity and nsitivity. It is recommended for targets up to 4 kb.
• Platinum ® Taq DNA Polymera High Fidelity provides incread fidelity and higher yields for targets up to 15 kb. •
Platinum ® Pfx DNA Polymera posss a proofreading 3´ to 5´ exonuclea activity and provides maximum fidelity for PCR. It is recommended for targets up to 12 kb.
Consult the product documentation provided with each DNA polymera for
recommended protocols and optimization guidelines. Documentation is also
available on our Web site at .
Page 3 of 4
Control Reactions
The control RNA provided with this system consists of total HeLa RNA (10 ng/µl). The n and antin control primers provided with this kit are designed from the human β-actin gene and produce a 353-bp RT-PCR product.
Sen primer: 5'-GCTCG TCGTC GACAA CGGCT C-3'
Antin primer: 5'-CAAAC ATGAT CTGGG TCATC TTCTC-3' U the following protocol for both plus and minus RT control reactions: 1. Dilute the total HeLa RNA to 100pg/µl with DEPC-treated water. 2.
Prepare the RNA/primer mixtures in sterile 0.2- or 0.5-ml tubes as follows:
Component + RT Control – RT Control Diluted total HeLa RNA (100 pg/µl) 1 µl 1 µl
Oligo(dT)20 1 µl 1 µl
10 mM dNTP mix 1 µl 1 µl
DEPC-treated water 7 µl 7 µl 3.
Incubate samples at 65°C for 5 min, then place on ice for at least 1 min.
Collect by brief centrifugation and add the following:
Component + RT Control – RT Control 10X RT buffer 2 µl 2 µl 25 mM MgCl 2 4
µl 4 µl 0.1 M DTT 2 µl 2 µl
RNaOUT ™ (40 U/µl) 1 µl 1 µl
SuperScript ™ III RT (200 U/µl) 1 µl —
DEPC-treated water — 1 µl 4. Mix gently and collect the reactions by brief centrifugation. 5. Incubate at 50°C for 50 min.
6. Terminate the reactions at 85°C for 5 min. Chill on ice.
7. Collect the reactions by brief centrifugation. Add 1 µl of RNa H to
each tube and incubate for 20 min at 37°C.
8.
Prepare a PCR mixture for each control reaction. For each control reaction, add the following to a 0.2-ml tube sitting on ice:
Component Volume
DEPC-treated water 38.1 µl
10X PCR buffer minus Mg ++ 5 µl 50 mM MgCl 2 1.5 µl
10 mM dNTP mix 1 µl Control n primer (10 µM) 1 µl
Control antin primer (10 µM) 1 µl
cDNA from control RNA 2 µl
Taq DNA polymera (5 units/µl) 0.4 µl
final volume 50 µl 9. Mix the contents of the tube. Centrifuge briefly to collect the reaction components. 10. Place reaction mixture in preheated (94°C) thermal cycler. Perform an initial denaturation step: 94°C for 2 min. 11.
Perform 40 cycles of PCR:
D enature 94°C for 15 c A nneal 55°C for 30 c
E xtend 68–72°C for 1 min
Note: For slow-ramping thermal cyclers, follow manufacturer’s directions.
12. Upon completion, maintain reactions at 4°C.
13. Analyze 10 µl of each sample, using agaro gel electrophoresis and
ethidium bromide staining. A 353-bp band, corresponding to at least 25 ng of product, should be visible for the + RT Control sample. No band should be visible for the – RT Control sample.
我们走了一些弯路First Strand cDNA Synthesis of Transcripts with High GC Content
High-GC content mRNAs often contain stable intrinsic condary structures that can inhibit rever transcripta and/or primer annealing. Problems with RT-PCR due to this condary structure often can be overcome by increasing the volume and temperature of the RT reaction.
Note: For templates that require cDNA synthesis temperatures above 55°C, we recommend the ThermoScript ™ RT-PCR System (Catalog no. 11146-024). ThermoScript ™ RT supports cDNA synthesis up to 70°C.
This protocol is suitable for gene-specific or oligo(dT) primers, but not
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random hexamers. 1. Mix and briefly centrifuge each component before u.
2. Prepare the RNA/primer mixture in a sterile 0.5-ml tube as follows: Component Sample Control RNA
1 to 5 µg total RNA n µl —八寨沟
Control total HeLa RNA (10 ng/µl) — 1 µl
Oligo(dT) 20 (50 µM) or 2 µM GSP 1 µl 1 µl
搞笑相声10 mM dNTP mix 2.5 µl 2.5 µl DEPC-treated water to 25 µl to 25 µl
3. Incubate each sample at 65°C for 5 min and immediately transfer to
55°C.
4. Prepare the cDNA Synthesis Mix, adding each component in the indicated order. Component 1 Reaction 10 Reactions
DEPC-treated water 3 µl 30 µl 10X RT buffer 5 µl 50 µl
25 mM MgCl 2 10 µl 100 µl 0.1 M DTT 5 µl 50 µl
RNaOUT ™
Recombinant RNa Inhibitor 1 µl 10 µl
SuperScript ™
III RT 1 µl 10 µl
Note: For a minus RT control reaction, substitute 1 µl of DEPC-treated water for 1 µl of SuperScript ™ III RT, and asmble reaction as described above.
5. Prewarm the cDNA Synthesis Mix to 55°C.
6. To each sample incubating at 55°C, add 25 µl of prewarmed cDNA
Synthesis Mix. Mix gently, and incubate at 55°C for 50 min. 7. Terminate the reactions at 85°C for 5 min. Chill on ice. 8. Collect the reactions by brief centrifugation. Add 1 µl of RNa H to each tube and incubate for 20 min at 37°C before proceeding to PCR. Note: Frequently, problems associated with RT-PCR of GC-rich cDNA are related to PCR as well as first-strand synthesis. We recommend using the PCR x Enhancer System (Catalog no. 11495-017) to facilitate amplification of GC-rich quences.
Page 4 of 4 Troubleshooting Guide
Problem Possible Cau Probable Solution
No bands after electrophoretic analysis of amplified products Procedural error in first-strand cDNA
synthesis
U the total HeLa RNA provided as a control to verify the efficiency of the
first-strand reaction (e page 3).
RNa contamination Add control RNA to sample to determine if RNa is prent in the first-strand
reaction.
Maintain aptic conditions to prevent RNa contamination.
U RNaOUT™ Recombinant RNa Inhibitor in the first-strand reaction. Polysaccharide coprecipitation of RNA Precipitate RNA with lithium chloride to remove polysaccharides, as described
in Sambrook et al.
Target mRNA contains strong
transcriptional paus
U random hexamers instead of oligo(dT) in the first-strand reaction.
Maintain an elevated temperature after the annealing step, as described in the
protocol for cDNA synthesis from high-GC content transcripts, page 3.
Increa the temperature of first-strand reaction (up to 55°C).
U PCR primers clor to the 3´ terminus of the target cDNA.
Too little first-strand product was ud in
PCR
U up to 10 µl of the first-strand reaction.
GSP was ud for first-strand synthesis Try another GSP or switch to oligo(dT). Make sure the GSP is the antin
quence.
Inhibitors of RT prent Remove inhibitors by ethanol precipitation of mRNA preparation before the
first-strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet.
Note: Inhibitors of RT include sodium dodecyl sulfate (SDS), EDTA,
guanidinium salts, formamide, sodium pyrophosphate, and spermidine.
Unexpected bands after electrophoretic analysis Contamination by genomic DNA Pretreat RNA with DNa I, Amplification Grade (Cat. no. 18068-015), as
described in the DNa I documentation.
手机证件照怎么拍Design primers that anneal to quence in exons on both sides of an intron or at
the exon/exon boundary of the mRNA to differentiate between amplified
cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, perform the minus RT control. Nonspecific annealing of primers Vary the annealing conditions. U Platinum®Taq DNA Polymera for
automatic hot-start PCR.
Optimize magnesium concentration for each template and primer combination. Primers formed dimers Design primers without complementary quences at the 3´ ends.
References
Berger, S.L. and Kimmel, A.R. (1987) Methods Enzymol 152, 316.
Bracete, A.M., Mertz, L.M., Fox, D.K. (1999) Focus® 21, 38.
Chomczynski, P. (1993) Biotechniques Vol. 15, 532.
Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
Compton, T. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 39, Academic Press, Inc.
Frohman, M.A., Dush, M.K, and Martin, G.R. (1988) Proc. Nat. Acad. Sci USA 85, 8998.
Gerard, G.F. (1994) Focus® 16, 102.
Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993) Focus® 15, 99.
Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus® 19, 46.
Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999) Focus® 21, 49.
Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol. 63, 4504.
Sitaraman, K., Darfler, M., and Westfall, B. (1999) Focus® 21, 10.
Nathan, M., Mertz, L., Fox, D. (1995) Focus® 17, 78.
Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J., Ronthal, K., Rashtchian, A., Gerard, G.F. (1998) Focus® 20, 30.
Limited U Label Licen No. 4: Products for PCR that include no rights to perform PCR
This product is optimized for u in the Polymera Chain Reaction (PCR) covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche, Ltd. (“Roche”). No licen under the patents to u the PCR process is conveyed expressly or by implication to the purchar by the purcha of this product. A licen to u the PCR process for certain rearch and development activities accompanies the purcha of certain reagents from licend suppliers such as Invitrogen, when ud in conjunction with an Authorized Thermal Cycler, or is available from
Applied Biosystems. Further information on purchasing licens to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.
Limited U Label Licen No. 138: SuperScript™ III Rever Transcripta
The purcha of this product conveys to the buyer the non-transferable right to u the purchad amount of the product and components of the product in rearch conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot ll or otherwi transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwi u this product or its components or materials made using this product or its components for Commercial Purpos. The buyer may transfer information or materials made through the u of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpo, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to u such transferred materials and/or information solely for rearch and not for Commercial Purpos. Commercial Purpos means any activity by a party for consideration and may include, but is not limited to: (1) u of the product or its components in manu
facturing; (2) u of the product or its components to provide a rvice, information, or data; (3) u of the product or its components for therapeutic, diagnostic or prophylactic purpos; or (4) resale of the product or its components, whether or not such product or its components are resold for u in rearch. Invitrogen Corporation will not asrt a claim against the buyer of infringement of patents owned by Invitrogen Corporation and claiming this product bad upon the manufacture, u or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in rearch by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was ud in the manufacture of such product. If the purchar is not willing to accept the limitations of this limited u statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a licen to this product for purpos other than rearch, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
Limited U Label Licen No. 18: RNaOUT™ Ribonuclea Inhibitor
This product is the subject of U.S. Patent No. 5,965,399 owned by Invitrogen Corporation. The purcha of this product conveys to the buyer the non-transferable right to u the purchad amount of the product and components of the product in rearch conducted by the buyer (whether t
he buyer is an academic or for-profit entity). The buyer cannot ll or otherwi transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwi u this product or its components or materials made using this product or its components for Commercial Purpos. The buyer may transfer information or materials made through the u of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpo, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to u such transferred materials and/or information solely for rearch and not for Commercial Purpos. Commercial Purpos means any activity by a party for consideration and may include, but is not limited to: (1) u of the product or its components in manufacturing; (2) u of the product or its components to provide a rvice, information, or data; (3) u of the product or its components for therapeutic, diagnostic or prophylactic purpos; or (4) resale of the product or its components, whether or not such product or its components are resold for u in rearch. Invitrogen Corporation will not asrt a claim against the buyer of infringement of the above patents bad upon the manufacture, u or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in rearch by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was ud in the manufacture of such product. If the purchar is not willing to accept the limitations of this limite
d u statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a licen to this product for purpos other than rearch, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
