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ABMG236
EasyScript Plus cDNA Synthesis Kit 100 reactions Protocol

EasyScript Plus cDNA Synthesis Kit 100 reactions <font color="blue">Protocol</font>
EasyScript Plus cDNA Synthesis Kit 100 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
recommended.
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
1 µl
Variable
 0.5 µM
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
RNaseOFF 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.

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