Takara Efficient preparation of cDNA: PrimeScript Reverse Transcriptase

Overview

• Strong strand-displacement and extension capability: capable of synthesizing long and full-length cDNA molecules (up to 12 kb)
• Preparation of cDNA at 42°C results in lower background and higher cDNA yields because the lower temperature does not promote RNA degradation
• Outstanding accuracy: lowest error rate among five commercially available reverse transcriptases tested
• Highly specific: low rates of non-specific annealing, even on incompletely denatured RNA
• Works on challenging templates: excellent results even with GC-rich templates and templates with high levels of secondary structure
• Highly sensitive: use less of your precious RNA samples
• Demonstrated success with reverse transcription reaction times of 30 min (60 min for longer transcripts)

More Information

Applications

• RT-PCR
• First-strand cDNA synthesis
• cDNA probe preparation
• Synthesis of cDNA libraries with a high proportion of full-length cDNAs

Standard protocol

1. Prepare the following mixture in a microtube.

|     |     |
| --- | --- |
| Oligo dT primer\* | 50 pmol |
| dNTP Mixture (10 mM each) | 1 µl |
| Template RNA | ≤5 µg (total RNA) or ≤1 µg (mRNA) |
| RNase-free dH2O | to 10 µl |


\* For Random Primers (6-mers), use 50 pmol. For gene-specific primers, use 2 pmol.

2. Heat at 65°C for 5 min, then immediately cool on ice.
3. Prepare the reaction mixture by combining the following components to create a 20-µl reaction.

|     |     |
| --- | --- |
| Template RNA/Primer Mixture | 10 µl |
| 5X PrimeScript Buffer | 4 µl |
| RNase Inhibitor | 20 U |
| PrimeScript Reverse Transcriptase | 100 U |
| RNase-free dH2O | to 20 µl |

4. Mix gently.
5. Perform the reaction under the following conditions.

|     |     |
| --- | --- |
| 30°C | 10 min\* |
| 42°C\*\* | 30–60 min |


\* This step is required for random primers.  
\*\* PrimeScript RTase has robust extension capability even with template having complex secondary structure. Therefore, incubation at 42°C is generally recommended. However, for RT-PCR reactions where the reverse primer for PCR is also used as a reverse transcription primer, we recommend performing the reverse transcription reaction at 50°C for 30 min to reduce the possibility of non-specific amplification products.

6. Heat at 70°C for 15 min and cool on ice.
   The resulting product can be used for a second-strand synthesis reaction or as a template for PCR.

Components

• Cat. # 2680A:

PrimeScript Reverse Transcriptase	10,000 U
5X PrimeScript Buffer	500 µl

• Buffer composition

5X PrimeScript Buffer (for cDNA synthesis)
50 mM	Tris-HCl, pH 8.3
375 mM	KCl
15 mM	MgCl2

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 mM: 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

Storage buffer composition

• 20 mM Tris-HCl, pH 7.8
• 100 mM NaCl
• 1 mM EDTA
• 1 mM DTT
• 50% Glycerol (v/v)

Choosing a PrimeScript kit for endpoint RT-PCR

If you want to...	Use this...
Prepare full-length cDNA using your own primers	PrimeScript Reverse Transcriptase (Cat. # 2680A and 2680B)
Prepare full-length cDNA using a kit that includes primers	PrimeScript First Strand cDNA Synthesis Kit (Cat. # 6110A and 6110B)
Perform 2-step RT-PCR with a kit that includes RT, primers, and PCR enzyme	PrimeScript RT-PCR Kit (Cat. # RR014A and RR014B)
Perform cDNA cloning studies with extremely high fidelity	PrimeScript High Fidelity RT-PCR Kit (Cat. # R022A and R022B)
Perform 1-step PCR	PrimeScript One-Step RT-PCR Kit, Ver. 2 (Cat. # RR055A and RR055B)

Additional product information

Please see the product`s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.