1x1 image pixel for data collection
SuperScript™ III First-Strand Synthesis SuperMix
For superior performance upgrade to the SuperScript IV First-Strand Synthesis System
SuperScript™ III First-Strand Synthesis SuperMix
Invitrogen™

SuperScript™ III First-Strand Synthesis SuperMix

The SuperScript III First-Strand Synthesis SuperMix is an optimized formulation for first-strand cDNA synthesis from purified poly(A)+ or total RNA.Read more
Have Questions?
Catalog NumberQuantity
1808040050 rxns
Catalog number 18080400
Price (USD)
673.65
Online Exclusive
744.00
Save 70.35 (9%)
Each
-
Add to cart
Quantity:
50 rxns
Recurring order eligible. Learn more »
Request bulk or custom format
Price (USD)
673.65
Online Exclusive
744.00
Save 70.35 (9%)
Each
Add to cart
Ask our AI about this Product

The SuperScript III First-Strand Synthesis SuperMix is an optimized formulation for first-strand cDNA synthesis 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 0.1 pg to 5 μg of total RNA.

This kit includes SuperScript III/RNaseOUT enzyme mix, 2X First-Strand reaction mix, and an annealing buffer. SuperScript III Reverse Transcriptase is a version of MMLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 45–55°C, providing increased specificity, higher yields of cDNA, and more full-length product than many previous reverse transcriptases.

Using SuperScript III First-Strand Synthesis SuperMix
SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA and can be used to synthesize cDNA from total RNA. RNaseOUT Recombinant RNase Inhibitor is included in the enzyme mix to safeguard against degradation of target RNA due to ribonuclease contamination. The 2X First-Strand reaction mix includes 10 mM MgCl2 and 1 mM of each dNTP in a buffer formulation that has been optimized for first-strand synthesis of cDNA. The annealing buffer is used in the initial template-primer annealing step. Separate tubes of oligo(dT)20 and random hexamers are also provided. cDNA synthesis can be performed using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer.

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Final Product TypeFirst-Strand cDNA
FormatKit
No. of Reactions50 Reactions
Optimal Reaction Temperature50°C
PrimerRandom Primers, Oligo dT Primers
Quantity50 rxns
Reaction FormatMaster Mix
Reagent TypeReverse Transcription
Reverse TranscriptaseSuperScript III
Shipping ConditionDry Ice
Starting MaterialRNA
TechniqueReverse Transcription
For Use With (Application)Real Time PCR (qPCR)
GC-Rich PCR PerformanceHigh
Reaction Speed50 min.
Unit SizeEach
Contents & Storage

• 2X Reaction Mix, 500 μL
• SuperScript™ III Enzyme Mix, 100 μL
• Oligo(dT)20, 50 μL total (50 μM)
• Random hexamers, 50 μL (50 ng/μL)
• Annealing buffer, 50 μL

Store at –20°C.

Have questions about this product? Ask our AI assisted search.
The DTT in my reverse transcription kit has precipitated—can I still use it?
I'm setting up my RT reaction and am trying to decide whether I should use random primers, oligo(dT) primer, gene-specific primer, or oligo(dT)/random mix primers. What would you suggest?
How much RNA should be employed for first-strand cDNA synthesis?
What is the difference between SuperScript III RT and the RT in the SuperScript VILO kit?
Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse transcription with SuperScript RT?
+Show more FAQs for this product
This is an AI-powered search and may not always get things right. You can help us make it better with a thumbs up or down on individual answers or by selecting the “Give feedback" button. Your search history and customer login information may be retained by Thermo Fisher and processed in accordance with our Privacy Notice.

Figures

Customers who viewed this item also viewed



Documents & Downloads

Certificates

Lot #Certificate TypeDateCatalog Number(s)
3139920Certificate of AnalysisMar 31, 202518080400
3040502Certificate of AnalysisJan 29, 202518080400
2984184Certificate of AnalysisSep 04, 202418080400
2851372Certificate of AnalysisApr 30, 202418080400
2525870Certificate of AnalysisFeb 21, 202418080400
5 results displayed, search above for a specific certificate

Safety Data Sheets

Frequently asked questions (FAQs)

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

If amplification products are generated in the control tube/well that contains no reverse transcriptase (i.e., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions. Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:
1.Total RNA, ideally, less than or equal to 1 µg. (See Note 1 below.)
2.1.0 µL of 10X DNase buffer (200 mM Tris, pH 8.3, 500 mM KCl, 20 mM MgCl2).
3.0.1 U-3.0 U of DNase I (RNase-free, Cat. No. 18047019) or 1.0 U Dnase I, Amplification Grade (Cat. No. 18068015. (See Note 2 below.)
4.Bring volume up to 10 µL with DEPC-treated water.
5.Incubate at room temperature for 15 min. (See Note 3 below.)
6.Terminate the reaction by adding 1 µL 25 mM EDTA and heat 10 min at 65 degrees C. (See Note 4 below.)
7.Place on ice for 1 minute.
8.Collect by brief centrifugation. This mixture can be used directly for reverse transcription.

Please note the following:
1.To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 µg RNA in the 10 µL reaction. More RNA will increase the viscosity of the solution and prevent the DNAse I from diffusing and finding the DNA.
2.DNAse I, Amplification Grade has been extensively purified to remove trace ribonuclease activities commonly associated with other "RNAse-free" enzyme preparations and does not require the addition of placental RNAse inhibitor.
3.It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg2+-dependent hydrolysis of the RNA.
4.This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg2+) is controlled.
5.Because the DNAse I must be heated to 65 degrees C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.
After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg2+ :EDTA. EDTA chelates Mg2+ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg2+. If the reverse transcription buffer does not contain MgCl2, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl2.

References on RNA hydrolysis:
Molekulyarnaya Biologiya (1987) 21:1235-1241.
References on the mechanism of hydrolysis by other cations:
Eichorn GL and Butzov JY (1965) Biopolymers 3:79.
Butzov JY and Eichorn GL (1965) Biopolymers 3:95.
Farkas WR (1968) Biochim Biophys Acta 155:401.
The authors of the first paper express the opinion that the mechanism of the nonspecific hydrolysis by cations which proceeds through 2',3' cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.

The amount of RNA template for a cDNA synthesis is highly flexible and depends upon the amount of sample available and an individual's need. In general, 1 µg total RNA is used in a typical 20-µL RT reaction.

Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

Some feel that the RNA in the RNA:DNA duplex after reverse transcription will inhibit PCR primers from annealing and amplifying the cDNA. The RNA is still present when using RNase H-mutant RTs. RNase H frees the cDNA from the RNA. On the other hand, some feel that the 95 degrees C denaturing step will cause the RNA primers to fall off the DNA and therefore RNase H treatment is not necessary. Therefore, this step is optional. For cloning of larger fragments, RNase H treatment can be beneficial.

This depends highly on the quality of the sample. mRNA itself makes up 1-5% of total RNA. Depending on the primer and enzyme used, reverse transcription can covert >70% of that into cDNA.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

Citations & References (4)

Citations & References
Abstract
TDAG51 is an ERK signaling target that opposes ERK-mediated HME16C mammary epithelial cell transformation.
Authors:Oberst MD, Beberman SJ, Zhao L, Yin JJ, Ward Y, Kelly K,
Journal:BMC Cancer
PubMed ID:18597688
'INTRODUCTION: Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ... More
Elevated serum IL-10 levels in diffuse large B-cell lymphoma: a mechanism of aberrant JAK2 activation.
Authors:Gupta M, Han JJ, Stenson M, Maurer M, Wellik L, Hu G, Ziesmer S, Dogan A, Witzig TE,
Journal:Blood
PubMed ID:22323454
'Cytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in ... More
Interactive effects of neurohypophyseal neuropeptides with receptor antagonists on passive avoidance behavior: mediation by a cerebral neurohypophyseal hormone receptor?
Authors:de Wied D, Elands J, Kovács G,
Journal:Proc Natl Acad Sci U S A
PubMed ID:1847526
The neurohypophyseal neuropeptides (Arg8)-vasopressin (AVP) and [pGlu4,Cyt6]AVP-(4-8) (where pGlu is pyroglutamic acid and Cyt is cystine) facilitate the retention of one-trial-learning passive avoidance behavior in rats when administered into the cerebral ventricle immediately after the learning trial. The fragment [pGlu4,Cyt6]AVP-(4-8) was considerably more effective than AVP. Oxytocin (OXT) and [pGlu4,Cyt6]OXT-(4-8) ... More
Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication.
Authors:McLean JE, Wudzinska A, Datan E, Quaglino D, Zakeri Z,
Journal:J Biol Chem
PubMed ID:21511946
Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely ... More
4 total citations

Other products to consider



Share catalog number, name or link