Product Datasheet
 
Product Description
 
Product: Reality Reverse Transcriptase cDNA Synthesis Kit
Catalog Number: CDNA
Product Page: https://www.mol-innov.com/products/IHUCCL21LY100REACTIONS
Description: Reality Reverse Transcriptase is a modified version of M-MLV reverse transcriptase with increased thermal stability and deactivated RNase H activity. In combination with optimized buffers and reagents, Reality Reverse Transcriptase synthesizes complementary DNA from single-stranded RNA or DNA templates. The enzyme is sensitive, specific and capable of synthesizing highly-structured and long cDNA fragments. The cDNA produced is appropriate for both PCR and qPCR analysis. Reality offers higher cDNA yields than some leading competitors with highly sensitive efficient synthesis from low-abundance RNA transcripts. Suitable for cDNA Synthesis of products from 100bp up to 10kb. The kit contains Reality Reverse Transcriptase (200U/µl), 5X buffer, dNTP mix (10mM), DTT solution (100mM), RNase inhibitor (40U/µl), Oligo dT 20mer primer (100µM), random hexamers (100µM), positive control RNA (10ng/µl), and RNase-free water.

Protocol: The following setup is recommended for a 20µl reaction.

Step 1A: cDNA synthesis without denaturation
ComponentStock ConcentrationFinal ConcentrationVolume
RNA Template-Total RNA: 10pg-5µg or mRNA: 10pg-500ngX µl
Primer100µMGene Specific Primer: 10-20pmol (50-100ng)
Oligo dT or random hexamer: 50pmol		0.1-0.2µl
0.5µl
dNTP Mix10mM500µM1µl
DTT Solution100mM5mM1µl
RNase Inhibitor40U/µl40U1µl
Reality Reverse Transcriptase200U/µl100U0.5µl
5X Buffer5X1X4µl
RNase Free Water--QS to 20µl

Step 1B (Optional): cDNA synthesis with denaturation
First, prepare the Template/Primer mix using the table below and incubate 5min at 65-70°C.
ComponentStock ConcentrationFinal ConcentrationVolume
RNA Template-Total RNA: 10pg-5µg or mRNA: 10pg-500ngX µl
Primer100µM Gene Specific Primer: 10-20pmol (50-100ng)
Oligo dT or random hexamer: 50pmol		0.1-0.2µl
0.5µl
RNase Free Water--QS to 10µl

Second, prepare the Reaction mix using the table below.
ComponentStock ConcentrationFinal ConcentrationVolume
dNTP Mix10mM500µM1µl
DTT Solution*100mM5mM1µl
RNase Inhibitor**40U/µl40U1µl
Reality Reverse Transcriptase***200U/µl100U0.5µl
5X Buffer5X1X4µl
RNase Free Water--QS to 10µl

*Adding up to 5mM DTT may increase the yield and is recommended for individual optimization.
**An additional 20-40 Units of RNase inhibitor per reaction is recommended when using low amounts of starting RNA.
***Increased transcription levels may be achieved by increasing the amount of enzyme to 200 Units.

Third, add 10µl of Reaction mix to 10µl of Template/Primer mix and pipette gently up and down on ice.

Step 2: Incubation
Gene Specific Primers: 30-60min at 50°C.
Oligo dT or random hexamer: 10min at 42°C followed by 30-60min at 50°C.

Step 3 (Optional): Heat inactivation
Heat the mixture for 10min at 70°C to inactivate Reverse Transcriptase.

Step 4 (Optional): RNA removal
Add 2 Units of DNase-free RNase and incubate for 20min at 37°C. The cDNA can now be used as a template in PCR and should be stored at -20°C.
Recommended Storage: SAMPLE
Minimum Shelf Life: 422.75
Molecular Weight: Proteins
Antigen: -70 C
Target: Complete kit
Target Molecular Weight: MOLECULAR BIOLOGY REAGENTS
Host: 817
Clonality: 5
Isotype: E. coli
Applications: Dry ice
Concentration: 445
Volume: 860
Buffer: January, 2016
Form: 100 reactions
 
Optimal experimental conditions and working dilutions must be determined by the end user. Sample concentration and volume are for example purposes only and do not necessarily represent the product that will be received. Please contact us for a current lot-specific product datasheet.
 
For in vitro laboratory use only
 
Molecular Innovations, Inc. - 46430 Peary Court, Novi, MI 48377, USA
Tel: (888) 557-5055 - Fax: (248) 896-0148 - info@mol-innov.com