Abbexa's cDNA Synthesis Kit allows rapid and very sensitive first strand cDNA synthesis for use in RT-qPCR applications. The Reverse Transcription Buffer contains a proprietary formulation designed for efficient reverse transcription and enhanced accuracy and includes all the necessary components for cDNA synthesis, including oligo dT and random hexamer primers, as well as an RNase inhibitor. The Reverse Transcriptase delivers highly robust first strand synthesis and high cDNA yields from a wide range of initial RNA concentrations.

Contents:

Component	50 rxns	250 rxns
5X Reverse Transcription Buffer	200 µl	1 ml
Reverse Transcriptase	50 µl	250 µl

Target	cDNA Synthesis Kit
Tested Applications	PCR
Form	Liquid
Storage	Store at -20 °C. Avoid repeated freeze/thaw cycles.
Validity	Up to 12 months.
Buffer	The exact formulation is proprietary.
Availability	Shipped within 3-7 working days.
Note	THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION.
Directions for use	Reaction Components: Prepare the master mix on ice. Vortex solutions and centrifuge briefly before use. Prepare a 20 µl reaction system according to the table below. Component Volume Total RNA or mRNA Variable 5X Reverse Transcription Buffer 4 µl Reverse Transcriptase 1 µl DNase/RNase-Free Water (not included with this product) Variable, up to 20 µl Total Volume 20 µl Mix gently by pipetting. Thermal Cycling Conditions: Step Temperature Time Primer Annealing 25 °C 10 min Reverse Transcription 42 °C 15 min Optional step recommended for highly-structured RNA such as viral RNA and some plant RNA 48 °C 15 min Inactivation 85 °C 5 min Keep the reaction mixture on ice (4 °C). Use up to 1/5th of the reaction volume (4 µl) of cDNA synthesis reaction product in a 20 µl reaction volume qPCR. If required, the reaction product can be diluted in a buffer consisting of 20 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA. If the reaction product is not immediately used in subsequent reactions, aliquot and store at 4 °C for up to 1 week or at -20 °C for long-term storage. Notes: Ensure that all reagents used are RNase-free. Reagents must be prepared using nuclease-free molecular biology grade water. Reverse transcriptase inhibitors, such as SDS, EDTA, formamide and pyrophosphate, should be removed by precipitating the RNA using ethanol, followed by a 70% ethanol wash step. If no cDNA synthesis is observed, the following adjustments may help: increase the amount of starting RNA; increase the primer annealing step time up to 15 min; increase the the reverse transcription step time up to 30 min. If the sample is contaminated with genomic DNA, treat samples with DNase I and repurify. If possible, use intron-spanning primers in qPCR. If non-specific annealing of primers to the template is observed, increase the annealing temperature in qPCR. The presence of pseudogenes may also contribute to non-specific annealing. If primer dimers are observed, ensure that reaction set-up is carried out on ice. The primers may need to be redesigned to prevent self-annealing.

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