New England Biolabs, E6560S, ProtoScript® II First Strand cDNA Synthesis Kit

The ProtoScript Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications cDNA Synthesis,, Reverse Transcription (cDNA Synthesis),, RNA Cloning, FAQ Q: How do I choose between LunaScript® RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits? A: The first strand cDNA synthesis kits contain different reverse transcriptases with the major differences being their primary applications, format, and reaction temperature. Here is a summary of our three first strand cDNA synthesis kits. Product LunaScript RT SuperMix Kit (E3010) ProtoScript II First Strand cDNA Synthesis Kit (E6560) ProtoScript First Strand cDNA Synthesis Kit (E6300) RT Luna reverse transcriptase ProtoScript II reverse transcriptase M-MuLV reverse transcriptase Format Supermix Reaction mix, enzyme mix, primer Reaction mix, enzyme mix, primer Primary Application Two-step RT-qPCR Endpoint RT-PCR up to 10kb Endpoint RT-PCR up to 5kb Secondary Application Endpoint RT-PCR up to 3kb Two-step RT-qPCR Two-step RT-qPCR Reaction Temperature Up to 65°C (55°C optimal) Up to 48°C (42°C optimal) Up to 45°C (42°C optimal) RNA Input 1μg to 1pg 1μg to 1pg 1μg to 1pg Incubation 25°C 2 min, 55°C 10 min, 95°C 1 min 25°C 5 min, 42°C 60 min, 85°C 5 min 25°C 5 min, 42°C 60 min, 85°C 5 min Additional Features Blue tracking dye, fast 13 min protocol Q: What is the difference between E6560 and E6300? A: E6560 includes M-MuLV H– reverse transcriptase in the enzyme mix, while E6300 includes wilde type M-MuLV reverse transcriptase in the enzyme mix. M-MuLV H– reverse transcriptase is a mutant M-MuLV Reverse transcriptase with reduced RNase H activity and increased thermostability. The enzyme is active up to 50C, providing higher specificity, higher yield of cDNA, and more full-length cDNA product. Q: What is the reaction temperature for E6560? A: We recommend 42C for routine reverse transcription. Up to 50C can be usedM-MuLV Reverse transcriptase (RNase H–) is capable of generating cDNA of more than 10kb up to 48C. Q: Is RNaseH treatment required before PCR amplification? A: For most RT-PCR reactions, RNaseH treatment is not required. But for some difficult amplicons or sensitive assays, add 2U of E.coli RNaseH to the reaction and incubate at 37°C for 20min. Q: Can the cDNA products be used in real-time PCR analysis? A: Yes. The cDNA products generated by ProtoScript II Reverse Transcriptase can be used directly for real-time PCR analysis. Q: What thermostable DNA polymerase can be used for PCR after cDNA synthesis? A: For downstream PCR, we recommend OneTaq 2X Master Mix (NEB #M0482 or M0485) for PCR detection up to 5kb, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for highest fidelity, or LongAmp Taq 2X Master Mix (NEB #M0287) for high yields from longer products. Q: How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products? A: Induro Reverse Transcriptase is a group II intron-encoded RT that is useful for cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. In contrast, the LunaScript reagents are master mixes. The LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) is suitable for general cDNA synthesis. LunaScript RT SuperMix products (NEB #E3010/M3010) are optimized for two step RT-qPCR workflows where only short cDNA products are required. LunaScript RT SuperMix is also featured in the ARTIC SARS-CoV-2 sequencing workflow. This table outlines the applications and features of each. Intron-encoded RT Retroviral RT Products Induro™ Reverse Transcriptase (NEB #M0681) LunaScript® RT SuperMix Kit (NEB #E3010) LunaScript RT SuperMix (NEB #M3010) LunaScript RT Master Mix Kit (NEB #E3025) ProtoScript® II First Strand cDNA Synthesis Kit (NEB #E6560) Applications Long cDNA synthesis Impure RNA samples RNA-seq applications (e.g., direct RNA sequencing, long-read cDNA sequencing, tRNA seq Applications 2-step RT-qPCR detection] RNA-seq workflow Applications RNA-seq workflow 2-step RT-qPCR detection General cDNA synthesis 2-step RT-PCR detection Applications General cDNA synthesis RNA-seq workflow 2-step RT-PCR detection ✓ Short protocol ✓ Flexible choice of primers, modified dNTPs ✓ Higher reaction temperature ✓ Supermix ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Kit format includes all necessary components Q: Why do I have low cDNA yields? A: There are several causes for low cDNA yield. Here are some suggestions to improve the yield: Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer (Agilent). Intact RNA of high purity is essential for full-length cDNA synthesis. RNA should have a minimum A260/A280 ratio of 1.7 or a RIN number greater than 8. Ethanol precipitation followed by a 70% ethanol wash can remove contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides. An RNA purification step using RNA extraction kits or phenol/chloroform extraction can remove contaminant proteins such as proteases. Increase the amount of RNA used, especially for low abundant RNA. Q: How do I know whether my template RNA is of good quality? A: Intact RNA of high purity is essential for full-length cDNA synthesis. An absorbance ratio at A260/A280 above 1.7 or RIN number greater than 8 on a BioAnalyzer usually indicates RNA of high quality.