New England Biolabs, E5315S, OneTaq® One-Step RT-PCR Kit

Easy and efficient One-Step RT-PCR Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications RT-PCR,, RT-PCR & cDNA Synthesis,, PCR FAQ Q: How do I choose between One-Step RT-PCR and Two-Step RT-PCR protocols? A: Both one-step and two-step RT-PCR protocols offer robust detection of RNA targets. Depending on your experimental design and goals, you may prefer one protocol over the other. Please refer to the following table: Q: What’s the difference between NEB #E6560 and NEB #E5315? A: Both kits use ProtoScript II Reverse Transcriptase for First Strand cDNA Synthesis. ProtoScript II Reverse Transcriptase is a mutant M-MuLV Reverse Transcriptase with reduced RNase H activity and increased thermostability. NEB #E6560 is a kit for first strand cDNA synthesis; its product can be used for downstream PCR or qPCR analysis. NEB #E5315 is a One-Step RT-PCR kit; its product is a specific dsDNA PCR product. Q: How does One-Step RT-PCR kit protocol work for NEB #E5315? A: The One-Step RT-PCR reactions allow both cDNA synthesis and PCR amplification in the same reaction buffer. A thermocycler is programmed so that cDNA synthesis takes place first (48°C for 15 minutes), then reverse transcriptase is inactivated while Hot Start Taq Polymerase is activated (94°C for 1 minute), followed by 40 cycles of PCR amplification. Q: Can I use a 25 μl reaction volume rather than a 50 μl reaction volume using NEB #E5315? A: Yes, you can use a 25 µl reaction by scaling down reagent volumes by half. Q: What conditions should I use for cDNA synthesis? A: We recommend using a cDNA synthesis temperature in the range of 42°C to 55°C for 15–30 minutes. Routinely, we use 48°C for 15 minutes. Q: Do I need to optimize the reaction components? A: The OneTaq One-Step RT-PCR Kit standard reaction conditions (1.6 mM MgCl2 and 250 µM dNTP at 1X) work well for most targets. You may be able to add more MgCl2 or dNTPs to make your reactions work better, but in most cases re-designing your primers may be more effective. Q: How much RNA template should I use? A: RNA templates can be total RNA, mRNA, or RNA synthesized by in vitro transcription. As a general rule, up to 1 μg of RNA template can be used in a 20 µl reaction. Q: How should I design my primers? A: We recommend following general guidelines for PCR primer design: primers are generally 20–40 nucleotides long, with GC content of 40–60%, and primer pairs should not share complementary sequences at the 3´ end. Q: What’s the recommended primer concentration? A: For most targets, primers at 200 nM to 400 nM work well. You may want to titer the primer concentration to the range of 100 nM to 800 nM. Q: How many PCR cycles should I do? A: We recommend 40 cycles for most targets. Some abundant RNA targets can be detected with as few as 25 cycles. Q: How do I choose between LunaScript Multiplex One-Step RT-PCR Kit and OneTaq One-Step RT-PCR Kit (NEB #E5315)? A: Both kits enable one-step RT-PCR. Please refer to the table below as a guideline to choosing between the LunaScript kit and the OneTaq One-Step RT-PCR Kit (NEB #E5315).