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EasyScript cDNA Synthesis SuperMix 25 Reactions Protocol

EasyScript cDNA Synthesis SuperMix 25 Reactions <font color="blue">Protocol</font>
EasyScript cDNA Synthesis SuperMix 25 Reactions

Product Description
EasyScript™ Reverse Transcriptase is an optimized mutational derivative of the original RTase
enzyme representing the best performing RTase on the market. This enzyme catalyzes the synthesis of complementary DNA strands from single-stranded RNA or DNA templates. Due to a series of mutations introduced within the RNase H domain of this enzyme, there is no detectable RNase H activity associated with the enzyme. The lack of RNase H activity helps to eliminate RNA degradation during first-strand cDNA synthesis, resulting in better yield and length of cDNA synthesized. Furthermore, EasyScriptTM RTase contains an additional fidelityenhancing subunit which drastically enhances accuracy in reverse transcription.

abm’s cDNA Synthesis Supermix is a proprietary mixture of all materials required for first-strand cDNA synthesis in a 2X concentration. This optimized reaction mix contains RNaseOFF Ribonuclease Inhibitor, dNTPs, and a balanced concentration for Oligo(dT) and Random Primers. RNaseOFF Ribonuclease Inhibitor effectively protects RNA template from degradation. Oligo(dT) anneals selectively to the poly(A) tail of mRNAs. Random Primers do not require the presence of poly(A) and they are utilized for the transcription of mRNA 5’-end regions. The first-strand cDNA can be directly used as a template in PCR.

Unit Definition

One unit is defined as the amount of enzyme required to incorporate 1 nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C using poly(A) and Oligo(dT) as template and primer, respectively.

Storage Buffer
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01 % (v/v) NP-40, 50 %
(v/v) glycerol.

Storage Conditions
Store all components at -20°C in a non-frost-free freezer. All components are stable for
1 year from the date of shipping when stored and handled properly.

Reverse transcription reactions should be assembled in a RNase-free environment. The use
of “clean”, automatic pipettes designated for PCR and aerosol-resistant barrier tips are

1. Thaw RNA templates and all reagents on ice. Mix each solution by vortexing gently.
2. Prepare the following reaction mixture on ice:
Components Volume Final Concentration
Total RNA or poly(A) + mRNA Variable 1 ng - 2 µg/rxn
1 pg - 2 ng/rxn
2X Reaction Mix 10 µl  1 X
Nuclease-free H2O  Up to 19 µl  -

3. Optional: Heat mixture to 65°C for 5 mins and incubate on ice for at least 1 min. Collect
all components by a brief centrifugation.
4. Add the following:
Components Volume Final Concentration
EasyScript™ RTase (200 U/µl)  1 µl  200 U/rxn

5. Mix components well and collect all components (20 μl) by a brief centrifugation.
Incubate the tube at 25°C for 10 mins. Perform cDNA synthesis by incubating the tube
for either 15 mins (for qPCR) or 50 mins (for PCR) at 42°C.
6. Stop the reaction by heating it at 85°C for 5 mins. Chill on ice. The newly synthesized firststrand
cDNA is ready for immediate downstream applications, or for long-term storage
at -20°C.

General Notes

1. Both poly(A) + mRNA and total RNA can be used for first-strand cDNA synthesis, but poly(A) + mRNA
may give higher yields and improved purity of final products.
2. RNA samples must be free of genomic DNA contamination.
3. Unlike Oligo(dT) priming, which requires little optimization, the ratio of Random Primers to RNA is
often critical in terms of the average length of cDNA synthesized. A higher ratio of Random Primers
to RNA will result in a higher yield of shorter (~500 bp) cDNA, whereas a lower ratio will lead to longer
cDNA products.
4. To remove RNA complementary to the cDNA, add 1 μl (2 U) of E. coli RNase H and incubate at 37°C
for 20 mins.

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