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Invitrogen™ SuperScript™ III First-Strand Synthesis SuperMix

Catalog No. 18080400
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Catalog No. 18080400 Supplier Invitrogen™ Supplier No. 18080400
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Description

Includes

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

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 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.

Order Info

Shipping Condition: Dry ice

Specifications

Content And 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.
Format Kit
GC-Rich PCR Performance High
Reaction Speed 50 min.
Technique Reverse Transcription
Optimal Reaction Temperature 50°C
Primer Random Primers, Oligo dT Primers
Quantity 50 rxns
Reverse Transcriptase SuperScript III
Shipping Condition Dry Ice
For Use With (Application) Real Time PCR (qPCR)
Final Product Type First-Strand cDNA
No. of Reactions 50 Reactions
Reaction Format Master Mix
Reagent Type Reverse Transcription
Starting Material RNA
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Frequently Asked Questions (FAQs)

How long can I store the cDNA from my reverse transcription step?

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.

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

We recommend using ezDNase (Cat. No. 11766051). ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55 degrees C). These features make ezDNase Enzyme an excellent choice for genomic DNA removal prior to reverse transcription reactions.
How much RNA should be employed for first-strand cDNA synthesis?

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.

Should I treat the cDNA with RNase H prior to downstream processing?

RNase H treatment is not always necessary. Many PCR reactions work without it. However, for cDNA synthesized with RNase H-deficient reverse transcriptases (like SuperScript II, III, and IV), RNA/cDNA hybrids—especially GC-rich ones—may not denature well, reducing PCR sensitivity. RNase H treatment can help in such cases. Additionally, RNase H treatment is beneficial for cloning larger fragments.

What percentage of RNA is converted to cDNA when performing reverse transcription?

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.
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?

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.

The DTT in my reverse transcription kit has precipitated—can I still use it?

No, the DTT will need to be replaced.

Are SuperScript II and III RTs RNase H minus?

These enzymes contain the domains of RNase H, but they have been mutated for reduced RNase H activity. In RNase H activity detection assays, we are not able to detect any RNase H activity.
Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse

EDTA chelates Mg2+ molecules on a 1:1 molar basis. If the amount of EDTA used for DNase I inactivation does not exceed the amount of Mg2+ present in the DNase reaction buffer, the resulting RNA solution can be used directly in the RT reaction. Otherwise, the sample should be purified to remove excess EDTA. Alternatively, consider using DNase removal methods that do not rely on EDTA or heat inactivation to avoid interference. To reduce the risk of compromised cDNA synthesis, we recommend performing gDNA removal with ezDNase Enzyme (Cat. No. 11766051) which is specific to dsDNA and heat-labile, hence does not require harsh inactivation conditions.
In comparing the different SuperScript III kit formats, I notice that some utilize a 10X buffer and others a 5X. The recipes are also slightly different - why is this?

It is recommended to use the buffer that comes supplied with the enzyme. The reasons for the slight differences are that the kits were developed at different times, possibly by different R&D groups.

Does SuperScript III exhibit TdT activity?

SuperScript III exhibits low TdT activity. If TdT activity is required, use our SuperScript IV RT or SuperScript IV Template Switching RT Master Mix.
What is the difference between SuperScript III RT and the RT in the SuperScript VILO kit?

The SuperScript VILO cDNA Synthesis Kit contains a mix of SuperScript III RT and helper proteins which help to increase the efficiency of the reverse transcription reaction and thus improve yield. The RT in the SuperScript VILO kit is active at 42 degrees C due to the helper proteins.

For Research Use Only. Not for use in diagnostic procedures.