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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 Plus™ Reverse Transcriptase is a novel recombinant reverse transcriptase that
exhibits much higher efficiency in the first-strand cDNA synthesis from RNA templates with
secondary structures and high GC content. The EasyScript PlusTM Reverse Transcriptase is
engineered to perform under high temperatures (50°C - 55°C), facilitating the elimination
of secondary structures associated with GC-rich RNA templates. Due to this unique feature,
EasyScript Plus™ can synthesize full-length cDNA libraries from RNA templates up to 15 kb
in length. In addition, EasyScript Plus™ Reverse Transcriptase has outstanding proofreading
ability due to the presence of a fidelity-enhancing subunit, thus making this RTase an excellent
choice for whole genome sequencing.

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 1X
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 PlusTM 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 50°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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