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Instruction manual ReverTra AceTM -α- 0810

ReverTra Ace -α-F0937K

TM
FSK-101F 100 reactions
Store at -20°C

Contents[1]

Introduction[2]

Components[3]

RT Primers[4]

Protocol
1. Template RNA for reverse transcription
2. Reverse transcription[5]

Applications for PCR[6]

Related protocol
DNase I treatment of total RNA[7]

Troubleshooting[8]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe
general laboratory precaution and utilize safety while using this kit.

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FOR RESEARCH USE ONLY.

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1

www.toyobo-global.com/seihin/xr/lifescience/[ 1 ] Introduction

Description
ReverTra Ace-α-TM is an efficient and convenient kit to synthesize high quality cDNA.
This kit contains the highly efficient reverse transcriptase “ReverTra AceTM”, as wellas other components optimized for the synthesis of long cDNAs suitable for RT-PCR.
ReverTra AceTM is an M-MLV reverse transcriptase that has been improved by point
mutation technology. ReverTra AceTM has two mutations in the polymerase and RNase
H domains.

Features-This kit contains all components for reverse transcription.
-This kit enables the synthesis of ≥ 14 kb cDNA.

[ 2 ] Components
The kit includes the following reagents which can be used for 100 reactions. All reagentsshould be stored at -20 °C.
ReverTra AceTM
5× RT buffer (contains 25 mM Mg2+)
RNase inhibitor (10 U/ µL)
dNTPs mixture (10 mM)
RNase-free H2O
Oligo (dT)20 (10 pmol/ µL)
Random primers (25 pmol/ µL)

100 µL400 µL100 µL200 µL
1200 µL
100 µL100 µL

Sequence of primers-Oligo (dT)20 :-Random Primer :JAPAN
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5’-(dT)20-3’5’-(dN)9-3’

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FOR RESEARCH USE ONLY.

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www.toyobo-global.com/seihin/xr/lifescience/[ 3 ] RT Primers

The following primers can be used for reverse transcription:
1. Oligo (dT) <Oligo (dT)20 primer is supplied by this kit>
This primer can be applied only to poly (A)+ RNA.
2. Random Primer <Random primer (9 mer) is supplied by this kit >This primer can be applied to various types of RNAs (e.g., total RNA, poly (A)+ RNA,tRNA, rRNA, and viral RNA). Because of the low Tm of this primer, the reverse
transcription reaction should include a pre-incubation step (30°C for 10 min.) to allow
for sufficient annealing to the template RNA.
3. Gene specific primer
Primers complimentary for mRNA can be used.

[4]Protocol

1.

Template RNA for reverse transcription

The following RNAs are appropriate for highly efficient reverse transcription:(1) Total RNA
Total RNA usually contains 1-2% mRNA and can be used directly as the template withthis kit. RNA prepared by AGPC (Acid Guanidinium-Phenol-Chloroform) or thecolumn method contains genomic DNA, so total RNA should be treated with DNase I
prior to transcription.
(2) Poly(A)+ RNA (mRNA)
Poly (A)+ RNA is useful for detecting low-level expressing mRNA. However, poly(A)+ RNA should be treated carefully, because Poly(A)+ RNA is more sensitive toRNase than total RNA.

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2.

Reverse transcription(1) Preparation of the reaction solutionComponent

Volume(11-X) µL

RNase-free H2O

Final concentration-5× RT buffer

4 µL

1×dNTP mixture (10 mM each)

2 µL

1 mM

RNase inhibitor (10 U/ µL)
PrimerRandom primer (25 pmol/ µL)
Oligo (dT)20(10 pmol/ µL)
Specific primer (10 pmol/ µL)
RNA
Total RNAmRNA [Poly (A)+ RNA]
ReverTra AceTM
Total Volume

1 µL
1 µL

0.5 U/ µL(2)

1.25 pmol/ µL
0.5 pmol/ µL
0.5 pmol/ µL
X µL≤ 1 µg10-100 ng
1 µL
20 µL

(Incubate at 30 °C for 10 min.) [In case of Random Primer only](3) Incubate at 42 °C for 20 min.
(4) Heat at 99 °C for 5 min.
(5) Store the reacted solution at 4°C or –20 °C
Notes-The heating step is necessary for dissociation of the DNA/RNA complex andincreased PCR efficiency-This kit contains RNase-free H2O for 100 reverse transcription reactions, but not forthe dilution of RNA samples. An RNase-free H2O, prepared without DEPC-treatment,is recommended for dilution of RNA samples.

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NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo-global.com/seihin/xr/lifescience/[ 5 ] Applicaionsfor PCR

The synthesized cDNA produced with this kit can be used as a template for efficient PCR
amplification. cDNA synthesis of ≥ 14 kb-targets have been confirmed.
It is known that residual RNA in cDNA or genomic DNA solutions inhibits
amplification through Mg2+ chelation. Therefore, PCR should be performed using atemplate DNA containing the following amounts of RNA, depending on each PCR
reagent:- KOD -Plus- (Code. KOD-201)- KOD FX (Code. KFX-101)- Blend Taq (Code. BTQ-101)- Blend Taq -Plus-(Code. BTQ-201)- KOD Dash (Code. LDP-101): ≤ 100 ng RNA/50 µL PCR reaction: ≤ 200 ng RNA/50 µL PCR reaction: ≤ 200 ng RNA/50 µL PCR reaction: ≤ 200 ng RNA/50 µL PCR reaction: ≤ 1 µg RNA/50 µL PCR reaction

PCR fidelity tends to decrease with excess amounts of Mg2+ or dNTPs; therefore, thereacted kit solution (≤ 2 µL) should be applied to the PCR solution for amplification withhigh fidelity enzymes, such as KOD -Plus- or KOD FX.

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www.toyobo-global.com/seihin/xr/lifescience/[ 6 ] Related
ProtocolDNase I treatment of total RNA
Total RNA prepared by general methods contains genomic DNA. Genomic DNA can beeliminated by the following method.
(1) Mix the following reagents.
Nuclease-free water

X µL

Total RNA (<10 µg)Y µL

10 x DNase I Buffer[e.g. 100 mM Tris-Cl, 20 mM MgCl2(pH 7.5)]

1 µL

RNase-free DNase I (10 U/ µL)

0.5 µL

Total volume

10 µL(2) Incubate on ice for 10-30 min.
(3) Purify the treated RNA according to the following step.
DNase I-treated RNA  Add nuclease-free water (adjust volume to 100 µL)
  Add 100 µL TE-saturated phenolVortexKeep on ice for 5 min.
 Centrifuge at 12,000 rpm for 5 min.
Supernatant  Add 100 µL chloroform: isoamyl alcohol (24:1), Vortex
 Centrifuge at 12,000 rpm for 5 min. at 4 °C
Supernatant Add 100 µL 5 M ammonium acetate + 200 µL isopropanol+ [5 µL 2 mg/mL glycogen* (for coprecipitation) : optional]
Vortex
Incubate at - 20 °C for 30 min.
 Centrifuge at 12,000 rpm for 10-15 min. at 4 °C
Discard supernatant
Precipitate Add 1 mL 70% ethanol Centrifuge at 12,000 rpm for 5 min.
Discard supernatant
Precipitate Dissolve in appropriate volume of nuclease-free water
RNA solution*Molecular biology grade
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www.toyobo-global.com/seihin/xr/lifescience/[ 7 ] Troubleshooting
SymptomLow efficiency

CauseSolution

Low purity of RNA

Re-purify the RNA sample.

RNA degradation

Prepare the RNA sample again.

Insufficient reaction

Prolong the reaction time up to 1 hr.

Excess or small amounts ofRNA

Optimal range of RNA template for reverse transcriptionis ≤ 1 µg of RNA.

Secondary structure of RNA-Use random primer.
-Incubate RNA solution at 65°C for 5 min., followed byincubation on ice prior to reaction.
Perform the reaction according to this instruction
manual.

Inappropriate temperature
conditions
Low specificity

Non-specific annealing of
gene-specific primers

Change the reaction temperature from 42°C to 50 °C.

[ 8 ] Relatedproducts
Product namePackage

Code No.

dNTPs mixture (10mM)

2 µmoles / 0.2 mL
10,000 U

NTP-301
TRT-1012,500 U200 reactions

SIN-201
FSQ-101

200 U

KOD-201

200 U

KFX-101

250 U

LDP-101

200 U

BTQ-101

200 U

BTQ-201

High efficient revers transcriptase
ReverTra AceTM

RNase inhibitor (Recombinant type)High efficient cDNA synthesis kit for real-time PCR
ReverTra AceTM qPCR RT Kit
High fidelity PCR enzyme (Hot Start)

KOD -PlusHigh reliable PCR enzyme (Hot Start)

KOD FX
High efficient DNA polymerase

KOD DashHigh efficient Taq DNA polymerase

Blend TaqHigh efficient Taq DNA polymerase (Hot Start)

Blend Taq -Plus-JAPANTOYOBO CO., LTD.
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CHINA
TOYOBO (SHANGHAI BIOTECH, CO., LTD.)
Tel(86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

6