PrimeScript Reverse

Transcriptase
Code No. 2680Q Purity
Shipping at 20 Nuclease activity is not detected in any of the following cases, as judged
Size: 2,000 units from the agarose gel electrophoresis pattern:
Store at 20 1. After incubation of 1 g of -Hin d III fragments with 200 units of this
enzyme for 1 hour at 37.
2. After incubation of 1 g of supercoiled pBR322 DNA with 200 units of
this enzyme for 1 hour at 37.
Supplied Reagents: 3. After incubation of 1 g of 16S and 23S rRNA with 200 units of this
5X PrimeScript Buffer 500 l enzyme for 1 hour at 37.

Composition of Supplied Reagent

Lot No. 5X PrimeScript Buffer (for cDNA synthesis )
250 mM Tris-HCl, pH8.3
Concentration: units/l 375 mM KCl
Volume: l 15 mM MgCl2

Expiration Date: Standard protocol for 1st-strand cDNA synthesis

1. Prepare the following mixture in a microtube.
Oligo dT primer 50 pmol
Description (or random primer (6 mers) 50 pmol )
PrimeScript Reverse Transcriptase is a modified M-MLV (Moloney Murine (or gene specific primer 2 pmol )
Leukemia Virus) RTase. This enzyme has extremely high extension dNTP Mixture (10 mM each) 1 l
capability and can synthesize long 1st-strand cDNA efficiently. Even for Template RNA total RNA 5 g, mRNA 1 g
difficult templates, including RNAs with complex secondary structure, it is RNase free dH2O up to 10 l
possible to synthesize 1st-strand cDNA at the normal reverse transcription 2. Heat at 65 for 5 min. and cool immediately on ice.
temperature (42) with this enzyme. It is not necessary to perform the 3. Prepare the reaction mixture by combining the following in a total
RT reaction at higher temperatures, a condition that may cause RNA volume of 20 l.
degradation. This enzyme is suitable for preparation of long cDNAs, Template RNA/Primer mixture 10 l
construction of cDNA libraries that include full-length cDNA, etc. 5X PrimeScript Buffer 4 l
RNase Inhibitor 20 units
Storage buffer PrimeScript Reverse Transcriptase 100 - 200 units 1
20 mM Tris-HCl, pH7.8 RNase free dH2O up to 20 l
100 mM NaCl 1: 100 units is recommended for RT-PCR and cDNA cloning; 200
1 mM EDTA units is recommended for quantitative analysis, such as qPCR.
1 mM DTT 4. Mix gently.
50% Glycerol (v/v) 5. Perform the reaction under the following condition.
30 10 min. 1
Source
42 ( - 50) 2 30 - 60 min. 3
Purified from an E. coli strain expressing a recombinant enzyme.
1: This step is required for random primer.
Unit definition 2: It is generally recommended to perform the RT reaction at 42.
One unit is the amount of the enzyme that incorporates 1 nmol of [3H] However, for RT-PCR, if the reverse primer for PCR is also used
dTTP in 10 minutes at 37, with poly (rA)oligo (dT)12-18 as the primer- as a RT primer, non-specific products may be amplified due to
template. mispriming. In such a case, perform the RT reaction at 50 for
30 min.
Reaction mixture for unit definition: 3: In most cases, 30 min. is sufficient. Increase the incubation time
50 m M Tris-HCl, pH8.3 to 60 min. when the target is very long.
75 mM KCl 6. Heat at 70 for 15 min. and cool on ice.
8 mM MgCl2
10 mM DTT The obtained cDNA can be used for 2nd-strand cDNA synthesis or as a
20 g/ml (rA)n(dT)12-18 template for PCR.
0.5 mM [3H] dTTP
0.1% NP-40
Note
1st-strand cDNA Synthesis Test This product is for research use only. It is not intended for use in
According to the standard protocol, 1st-strand cDNA was synthesized therapeutic or diagnostic procedures for humans or animals. Also, do
by incubating 100 ng of total RNA prepared from rat brain and 50 pmol not use this product as food, cosmetic, or household item, etc.
of Oligo dT primer with 200 units of PrimeScript Reverse Transcriptase at Takara products may not be resold or transferred, modified for resale
or transfer, or used to manufacture commercial products without
42 for 60 minutes. Then, PCR was performed using the RT reactant as a
written approval from TAKARA BIO INC.
template and amplification of a 12 kb target was confirmed by agarose gel If you require licenses for other use, please contact us by phone at
electrophoresis. +81 77 543 7247 or from our website at www.takara-bio.com.
Your use of this product is also subject to compliance with any
Applications applicable licensing requirements described on the product web
1. First-strand cDNA synthesis. page. It is your responsibility to review, understand and adhere to
2. Preparation of cDNA probes. any restrictions imposed by such statements.
3. RT-PCR. All trademarks are the property of their respective owners. Certain
trademarks may not be registered in all jurisdictions.
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 PrimeScript Reverse
Transcriptase
Code No. 2680Q
Shipping at 20 1. 200 U 1 g -Hin d III 371
Size: 2,000 units DNA
Store at 20 2. 200 U 1 g supercoiled pBR322 DNA 371
DNA
3. 200 U 1 g 23S 16S rRNA 37
1 RNA

5 PrimeScript Buffer 500 l 20

5 PrimeScript Buffer (cDNA )
250 mM Tris-HCl, pH8.3
Lot No. 375 mM KCl
15 mM MgCl2

1st-strand cDNA
1.
Oligo dT primer 50 pmol
(or Random primer (6 mers) 50 pmol )

(or Gene specific primer 2 pmol )

PrimeScript Reverse Transcriptase M-MLV (Moloney Murine Leukemia
dNTP mixture (10 mM each) 1 l
virus) Reverse Transcriptase
RNA total RNA5 g mRNA1 g

RNase free dH2O up to 10 l

cDNA GC cDNA
2. 65
RNA RNA
3. 20 l
(42) cDNA
RNA/Primer Mixture 10 l
cDNA cDNA
5 PrimeScript Buffer 4 l

RNase Inhibitor 20 units

PrimeScript Reverse Transcriptase 100 200 units
20 mM Tris-HCl, pH7.8 RNase free dH2O up to 20 l
100 mM NaCl 4.
1 mM EDTA 5.
1 mM DTT 30 10 min. 1
50% Glycerol (v/v) 42 ( 50) 2 30 60 min.
1: Random 6 mers
2: PrimeScript Reverse Transcriptase
42RT-PCR
PCR
Poly(rA)oligo(dT)12-18 3710
1 nmol [3H] dTTP 1 U 50

6. 70 15
50 mM Tris-HCl, pH8.3
75 mM KCl PCR 2nd-strand
8 mM MgCl2
10 mM DTT
20 g/ml (rA)n(dT)12-18
0.5 mM [3H]dTTP
0.1% NP-40

1st-strand cDNA
total RNA 100 ng

Oligo dT Primer 50 pmol 200 units 42 60

1st-strand cDNA cDNA

PCR 12 kb

1. 1st-strand cDNA

2. cDNA

3. RT-PCR

v201311Da

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Fax 077-543-1977