Protocol Pub. No. MAN0013443 Rev. A.

SuperScript® IV Reverse Transcriptase

Protocol outline
Catalog Number Size
Package Kit Contents
A. Anneal primer to RNA
18090010 2,000 units
contents B. Assemble reaction mix
Print Options

18090050 10,000 units

18090200 4 × 10,000 units C. Add reaction mix to annealed RNA

Storage RT reaction setup

Store at –20°C (non-frost-free)
conditions Use the measurements below to prepare your RT reaction, or enter your own
parameters in the column provided.
∤ Template: RNA Component 20-µL rxn Custom Final Conc.
Oligo(dT) primer (Cat. no. 18418-020),
∤ random 20
hexamers (Cat. no. N8080127), DEPC-treated water to 20 µL to µL N/A
or 2 μM gene-specific primers
Required 10 mM dNTP mix (Cat. no. 18427-013) 5× SSIV Buffer 4.0 µL µL 1×
materials
∤ 10 mM dNTP mix (10 mM each) 1.0 µL µL 0.5 mM each
∤ RNaseOUT™ Recombinant Ribonuclease Inhibitor
(Cat. no. 10777-019) 100 mM DTT 1.0 µL µL 5 mM
∤ E. coli Ribonuclease H (RNase H) (Cat. no. 18021-014) RNaseOUT™ RNase Inhibitor
DEPC-treated water (Cat. no. 10813-012) 1.0 µL µL 2.0 U/µL
∤ (40 U/µL)

∤ Preparation time: 10 minutes 50 µM Oligo d(T)20 primer, or 1.0 µL µL 2.5 µM

Timing 50 µM random hexamers, or 1.0 µL 2.5 µM
∤ Run time: 20 minutes 2 µM gene-specific primer 1.0 µL 0.1 µM
Go online to view related products. < 5 μg total RNA
PCR Enzymes and Master Mixes Template RNA* varies µL or
Selection RT Enzymes and Kits < 500 ng mRNA
guides Real-Time PCR Instruments * 10 pg–5 µg total RNA or 10 pg–500 ng mRNA
Real-Time PCR Master Mixes
PCR Thermal Cyclers
RT protocol
Go to page 2 for instructions on preparing and running your RT experiment.
For first strand cDNA synthesis using total RNA or
Product poly(A)+-selected RNA primed with oligo(dT), random Optimization strategies and troubleshooting
description primers, or a gene-specific primer.
Refer to the pop-ups below for guidelines to optimize and troubleshoot your
RT reaction.
Pre-warm the 5× SSIV Buffer to room temperature before use.
Important Vortex and briefly centrifuge the buffer prior to preparing the RNA Sample Prep RT Guidelines
guidelines reverse transcription reaction mix.
Troubleshooting
Online Visit our product page for additional information and protocols.
resources For support, visit [Link]/support.

Limited Warranty, Disclaimer,

and Licensing Information
For Research Use Only. Not for use in diagnostic procedures.
SuperScript® IV Reverse Transcriptase User Guide
SuperScript® IV First-Strand cDNA Synthesis Reaction
The example procedure below shows appropriate volumes for a single 20-µL reverse transcription reaction. For multiple reactions, prepare a master mix of components common
to all reactions to minimize pipetting error, then dispense appropriate volumes into each reaction tube prior to adding annealed template RNA and primers.

Steps Procedure Procedure details

a. Combine the following components in a reaction tube.
Note: Consider the volumes for all components listed in steps 1 and 2 to determine the correct
amount of water required to reach your final reaction volume.
Component Volume
50 µM Oligo d(T)20 primer, 50 µM random hexamers, or
Anneal primer to 1 µL
1 2 µM gene-specific reverse primer
template RNA 10 mM dNTP mix (10 mM each) 1 µL
Template RNA (10 pg–5 µg total RNA or 10 pg–500 ng mRNA) up to 11 µL
DEPC-treated or nuclease-free water to 13 µL
b. Mix and briefly centrifuge the components.
c. Heat the RNA-primer mix at 65°C for 5 minutes, and then incubate on ice for at least 1 minute.
a. Vortex and briefly centrifuge the 5× SSIV Buffer.
b. Combine the following components in a reaction tube.
Component Volume
5× SSIV Buffer 4 µL
2 Prepare RT reaction mix 100 mM DTT 1 µL
RNaseOUT™ Recombinant RNase Inhibitor 1 µL
SuperScript® IV Reverse Transcriptase (200 U/μL) 1 µL
c. Cap the tube, mix, and then briefly centrifuge the contents.

Combine annealed RNA

3 Add RT reaction mix to the annealed RNA.
and RT reaction mix

a. If using random hexamer, incubate the combined reaction mixture at 23°C for 10 minutes, and then
proceed to step b.
4 Incubate reactions If using oligo d(T)20 or gene-specific primer, directly proceed to step b.
b. Incubate the combined reaction mixture at 50–55°C for 10 minutes.
c. Inactivate the reaction by incubating it at 80°C for 10 minutes.

Note: Amplification of some PCR targets (>1 kb) may require removal of RNA.
5 Optional: Remove RNA
To remove RNA, add 1 µL E. coli RNase H, and incubate 37°C for 20 minutes.

Use your RT reaction immediately for PCR amplification or store it at –20°C.

6 PCR amplification Note: As a recommended starting point for PCR, reverse transcription reaction (cDNA) should
compose 10% of the total reaction volume

3 February 2015 -2- For support, visit [Link]/support.