b'Frequently asked questions Below are some common questions and answers to help you start or troubleshoot molecular biology experiments.Sample preparation What are the benefits of using random primers, oligo(dT) primers, Which kit should I use to isolate nucleic acids from my sample? gene-specific primers, or oligo(dT)/random mixed primers in Choosing the right product is fundamental to ensuring proper lysis of cellsreverse transcription?and tissue, as well as sufficient yield and quality of isolated nucleic acids. Random primers are good to use with degraded RNA, RNA with high Look to our selection guides (see pages 911) to help you decide accordingsecondary structure, nonpolyadenylated RNA, or prokaryotic RNA. to nucleic acid type, sample source, experimental throughput, and format as well as downstream applications.Oligo(dT) primers are an optimal choice for synthesis of full-length cDNA from eukaryotic mRNA. Applications include cDNA cloning, cDNA library What are the key steps to preventing RNA degradation?construction, and 3 rapid amplification of cDNA ends (3 RACE).The basic lab precautions listed below can help minimize RNA degradation Gene-specific primers are designed based on known sequences of and avoid experimental inconsistency and failure. the target RNA. These primers offer the most specific priming and areUse nuclease-free pipette tips and tubes commonly used in one-step RT-PCR.Use nuclease-free water and reagentsA mixture of oligo(dT) and random primers is often used in two-step RT-PCR to achieve the benefits of each primer type (see page 18).Regularly decontaminate work surfaces Properly stabilize RNA sources before storage For more tips and troubleshooting advice on reverse transcription, visit thermofisher.com/rteducation and thermofisher.com/rtsupportFor more tips and troubleshooting advice on sample prep, visitPCR amplificationthermofisher.com/rnabasics and thermofisher.com/napsupport How can I optimize primer annealing for PCR?Traditionally, gradient thermal cyclers have been used to simultaneously Reverse transcription assess a number of temperatures around the theoretical annealing point. How do I improve the efficiency of cDNA synthesis when workingCompared to gradient thermal cyclers, instruments with the VeriFlex with challenging samples (e.g., low-abundance, degraded, inhibitor- technology allow more precise temperature control for faster optimization of containing, or GC-rich RNA)? primer annealing (see page 21).When working with challenging RNA samples, select a reverse transcriptase that is highly sensitive, processive, thermostable, and resistant to commonTedious optimization steps may be circumvented using the novel Platinum inhibitors, to help you obtain the highest cDNA yield (see page 15). DNA polymerases. Their innovative buffers enable specific annealing at 60C for most primers when they are designed following general primer design rules (see pages 2829).Resources51'