SuperScript™ III Rever Transcripta
Cat. No. 18080-093 Size: 2,000 units
units
18080-044 10,000
× 10,000-unit kit
18080-085 4
Conc: 200 U/µl Store at -20°C (non-frost-free)
Description
SuperScript™ III Rever Transcripta is an engineered version of M-MLV
RT with reduced RNa H activity and incread thermal stability. The
enzyme is purified to near homogeneity from E. coli containing the
处处好风光modified pol gene of Moloney Murine Leukemia Virus (1,2). The enzyme
can be ud to synthesize first-strand cDNA at temperatures up to 55°C, providing incread specificity, higher yields of cDNA, and more full-
length product than other rever transcriptas. It can generate cDNA
from 100 bp to >12 kb.
Component 2,000 U Kit 10,000 U Kit
µl
µl 50 SuperScript™ III RT (200 U/µl) 10
µl
5X First-Strand Buffer* 1000 µl 1000
µl
0.1 M DTT 500 µl 500 *[250 mM Tris-HCl (pH 8.3 at room temperature), 375 mM KCl, 15 mM MgCl2]
Unit Definition
One unit incorporates 1 nmol of dTTP into acid-precipitable material in
10 min at 37°C using poly(A)•oligo(dT)25 as template-primer (3).
Storage Buffer
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01%
(v/v) NP-40, 50% (v/v) glycerol
Storage
Store all components at -20°C (non-frost-free). Thaw 5X First-Strand Buffer
and 0.1 M DTT at room temperature just prior to u and refreeze immediately.
Part no. 18080.pps Rev. date: 7 Dec 2004
For technical support, email tech_
For country-specific contact information, visit
Page 2 First-Strand cDNA Synthesis
The following 20-µl reaction volume can be ud for 10 pg–5 µg of total RNA or
10 pg–500 ng of mRNA.
1. Add the following components to a nuclea-free microcentrifuge tube:
1 µl of oligo(dT)20 (50 µM); or 200–500 ng of oligo(dT)12-18; or
50–250 ng of random primers; or 2 pmol of gene-specific primer
10 pg–5 µg total RNA or 10 pg–500 ng mRNA
1 µl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at
微信女生头像neutral pH)
Sterile, distilled water to 13 µl
2. Heat mixture to 65°C for 5 minutes and incubate on ice for at least 1 minute
3. Collect the contents of the tube by brief centrifugation and add:
4 µl 5X First-Strand Buffer
当今中国九大热点问题1 µl 0.1 M DTT
1 µl RNaOUT™ Recombinant RNa Inhibitor (Cat. no. 10777-019,
40 units/µl). Note: When using less than 50 ng of starting RNA, the
addition of RNaOUT™ is esntial.
1 µl of SuperScript™ III RT (200 units/µl)*
*If generating cDNA longer than 5 kb at temperatures above 50°C using a gene-specific primer or oligo(dT)20, the amount of SuperScript™ III RT may be raid to 400 U (2 µl) to increa yield.
4. Mix by pipetting gently up and down. If using random primers, incubate tube at
25°C for 5 minutes.
虎眼石5. Incubate at 50°C for 30–60 minutes. Increa the reaction temperature to 55°C for
gene-specific primer. Reaction temperature may also be incread to 55°C for difficult templates or templates with high condary structure.
6. Inactivate the reaction by heating at 70°C for 15 minutes.
The cDNA can now be ud as a template for amplification in PCR. However, amplification of some PCR targets (tho >1 kb) may require the removal of RNA complementary to the cDNA. To remove RNA complementary to the cDNA, add 1 µl (2 units) of E. coli RNa H and incubate at 37°C for 20 minutes.
Page 3
PCR Reaction
The following example reaction is recommended as a starting point:
1. Add the following to a PCR reaction tube:
杨孟衡10X PCR Buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl] 5 µl
50 mM MgCl2* 1.5
µl
10 mM dNTP Mix 1 µl
µl
Sen primer (10 µM) 1
µl
Antin primer (10 µM) 1
µl
Taq DNA polymera (5 U/µl) 0.4
cDNA (from first-strand reaction) 2 µl
Autoclaved, distilled water to 50 µl
中单
2. Mix gently and layer 1–2 drops (~50 µl) of silicone oil over the reaction.
(Note: The addition of silicone oil is unnecessary in thermal cyclers equipped with
a heated lid.)
3.Heat reaction to 94°C for 2 minutes to denature.
4. Perform 15–40 cycles of PCR. Annealing and extension conditions are
primer and template dependent and must be determined empirically.
*Optimal concentration of MgCl2 needs to be determined empirically for
each template-primer pair.
Quality Control
This product has pasd the following quality control assays: SDS-polyacrylamide
gel analysis for purity; functional abnce of endodeoxyribonuclea, 3′ and 5′
exodeoxyribonuclea, and ribonuclea activities; yield and length of cDNA
product.
References
1. Kotewicz, M.L., D'Alessio, J.M., Driftmier, K.M., Blodgett, K.P., and Gerard,
G.F. (1985) Gene 35, 249.
2. Gerard, G.F., D'Alessio, J.M., Kotewicz, M.L., and Noon, M.C. (1986) DNA 5,
271.
3. Houts, G.E., Miyagi, M., Ellis, C., Beard, A., and Beard, J.W. (1979) J. Virol.29,
517.
Page 4 Related Products
No.
Quantity Cat.
µl 18418-020 Oligo(dT)20 Primer (50 µM) 50
Oligo(dT)12-18 Primer 25 µg 18418-012 Random Primers A260 units 48190-011
Custom Gene-Specific Primers visit /oligos
10 mM dNTP Mix 100 µl 18427-013 DEPC-treated Water 4 × 1.25 ml 10813-012 RNAOUT™ Recombinant
units 10777-019 Ribonuclea Inhibitor (40 U/µl) 5,000
RNa H 30 units 18021-014
Platinum®Taq DNA Polymera 100 units 10966-018
Limited U Label Licen No. 4: Products for PCR which do not include any rights to
perform PCR
This product is optimized for u in the Polymera Chain Reaction (PCR) covered by patents owned by Roche Molecular
七夕节的诗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绘画鉴赏
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 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 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.
©2002–04 Invitrogen Corporation. All rights rerved.
For rearch u only. Not intended for any animal or human therapeutic or diagnostic u.
