Description

PrimeScript™ Reverse Transcriptase is a novel reverse transcriptase, developed by TaKaRa Bio Inc. based on RTase originated from M-MLV(Moloney Murine Leukemia Virus). Since this enzyme has extremely high extension-ability, it can synthesize long 1st-strand cDNA efficiently.
Even if the RNA, difficult of reverse-transcription due to its secondary structure, is used as a template, it is possible to synthesize the 1st strand cDNA at the normal reverse transcription temperature such as 42°C with this enzyme.
It is not necessary to perform the RT reaction at high temperature, which may cause RNA degradation.
This enzyme is suitable for preparation of long cDNA, construction of cDNA library including high proportion of full-length cDNA and so on.

This product is not available in the U.S.

Storage

- 20°C

Concentration

200 units/µl

Form

20 mM		Tris-HCl, pH7.8
100 mM		NaCl
1 mM		EDTA
1 mM		DTT
50 %		Glycerol (v/v)

Source

Purified from an E. coli strain expressing a recombinant enzyme.

Unit definition

One unit is the amount of the enzyme that incorporates 1 nmol of [³H]dTTP in 10 minutes at 37°C, with poly(rA),oligo(dT) _12-18 as the primer-template.

Reaction mixture for unit definition

50 m M		Tris-HCl, pH 8.3
75 mM		KCl
8 mM		MgCl2
10 mM		DTT
20 µg/ml		(rA)n·(dT)12-18
0.5 mM		[3H]dTTP
0.1 %		NP-40

1st-strand cDNA Extension Test

According to the standard protocol, 1st strand cDNA is synthesized by incubating 500 ng of total RNA prepared from mouse heart and 50 pmol of Oligo dT primer with 100 units of PrimeScript™ Reverse Transcriptase at 42°C for 30 minutes. Then, PCR is performed with this RT reactant as a template and it is confirmed that the 12 kbp target is properly amplified by agarose gel electrophoresis.

Purity

Nuclease activity is not detected in any of the following cases, as judged from the agarose gel electrophoresis pattern:

1.	After incubation of 1 µg of λDNA-HindIII fragments with 200 units of this enzyme for 1 hour at 37°C.
2.	After incubation of 1 µg of supercoiled pBR322 DNA with 200 units of this enzyme for 1 hour at 37°C.
3.	After incubation of 1 µg of 16S and 23S rRNA with 200 units of this enzyme for 1 hour at 37°C.

Application

1.First-strand cDNA synthesis.
2.Preparation of cDNA probe.
3.RT-PCR.

Composition of Supplied Reagent

5 × PrimeScript™ Buffer (for cDNA synthesis )

250 mM		Tris-HCl, pH 8.3
375 mM		KCl
15 mM		MgCl2

Standard protocol for 1st-strand cDNA synthesis

1. Prepare the following mixture in a microtube.

	Oligo dT primer	50 pmol
	(or Random primer (6 mers)	50 pmol)
	(or Gene specific primer	2 pmol)
	dNTP Mixture (10 mM each)	1 µl
	Template RNA	total RNA≤5 µg, mRNA≤1 µg
	RNase free dH2O	up to 10 µl

2. Heat at 65°C for 5 min. and cool immediately on ice.

3. Prepare the reaction mixture by combining the following reagents to a total volume of 20 µl.

	Template RNA/Primer mixture	10 µl
	5 × PrimeScript™ Buffer	4 µl
	RNase Inhibitor	20 units
	PrimeScript™ Reverse Transcriptase	100 - 200 units
	RNase free dH2O	up to 20 µl

4. Mix gently.

5. Perform the reaction under the following condition.

	30°C	10 min*
	↓
	42(- 50)°C **	30 - 60 min

* This step is required for random primer.
** PrimeScript™ Reverse Transcriptase shows powerful extension ability even if the template RNA has a strong secondary structure. Therefore, it is generally recommended to perform the RT reaction at 42°C with this enzyme. However, in case of RT-PCR, if the reverse primer for PCR is also used as a RT primer, non-specific products may be amplified due to mispriming. In such a case, it is recommended to perform the RT reaction at 50°C.

6. Heat at 95°C for 5 min.^*** and cool on ice.

*** For amplification of longer targets, the inactivation at 70°C for 15 min. is recommended, so there will be no damage to the 1st strand cDNA (i.e. nicking).

The obtained reactant can be used for 2nd strand synthesis reaction or as a template for PCR.