New England Biolabs, E6300S, ProtoScript® First Strand cDNA Synthesis Kit

The ProtoScript First Strand cDNA Synthesis Kit is contains everything needed for cDNA synthesis Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications cDNA Synthesis,, Reverse Transcription (cDNA Synthesis),, RT-PCR & cDNA Synthesis, 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: Why am I getting a low yield of cDNA? A: There are several possibilities: Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2). Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2). Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN. Use sufficient amount of RNA.