EasyScript Plus Reverse Transcriptase, 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 Plus™ 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. EasyScript PlusTM cDNA Synthesis Kit
contains all materials required for first-strand cDNA
synthesis, with the choice of using either Oligo(dT) and/or Random Primers. The Oligo(dT)
anneals selectively to the poly(A) tail of mRNAs. Random Primers do not require the presence
of poly(A) and can be used for the transcription of mRNA 5’-end regions. Gene specific
primers may also be used with the kit. The recombinant RNaseOFF Ribonuclease Inhibitor,
supplied with the kit, effectively protects RNA template from degradation. 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 37oC using poly(A) and Oligo(dT) as template
and primer, respectively. Primer Selection Oligo(dT)
are oligonucleotides that anneal to the 3’-poly(A) + mRNA. Therefore, only mRNA
or total RNA templates with 3’-poly(A) tails are used in cDNA synthesis. Random Primers
are oligonucleotides that anneal at non-specific sites of RNA templates.
Therefore, all forms of RNA can be used in cDNA synthesis. Gene-Specific
Primers are oligonucleotides that are designed to anneal to the specific site
of a target gene. 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. Protocol
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
|Oligo(dT) (10 µM) or Random Primers (10 µM) or Gene-Specific Primer || 1 µl |
| 0.5 µM |
10 - 15 nM
| dNTP Mix (10 mM each) || 1 µl || 500 µM |
| Nuclease-free H2O || Up to 14.5 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 |
| 5X RT Buffer ||4 µl || 1X |
| RNase OFF Ribonuclease Inhibitor (40 U/µl) || 0.5 µl || 20 U/rxn |
| 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 if using Random Primers. Omit this incubation if
Oligo(dT) or Gene-Specific Primer is used.
6. Perform cDNA synthesis by incubating the tube for either 15 mins (for qPCR) or 50 mins
(for PCR) at 50°C.
7. Stop 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. For laboratory research only. Not for clinical applications