New England Biolabs, E5310S, OneTaq® RT-PCR Kit

Easy and efficient two-step RT-PCR Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications RT-PCR,, RT-PCR & cDNA Synthesis,, Multiplex PCR, FAQ Q: Why do I have a low yield of PCR product? A: There are several things that may improve yields: Check the primer design using computer software. Optimize the annealing temperature in a 1-2°C step. A primer concentration of 0.2 μM is satisfactory for most PCR reactions. However, sensitivity and yield of RT-PCR reactions can be improved by increasing the primer concentration to above 0.5 μM. Lower primer concentration between 0.07 μM to 0.2 μM may improve specificity. Increase cycling numbers up to 45 cycles. Do a manual hot-start. Use thin-wall 0.2 ml PCR tubes. Try a touch-down PCR protocol (4). 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. Q: Why do I see products of the wrong size? A: There could be several reasons: The presence of a PCR product larger than expected is often due to the contamination of genomic DNA. Treat with DNase I prior to cDNA synthesis (5). The presence of a PCR product of the correct size in the -RT negative control is either due to contaminating genomic DNA or carryover PCR product. Use separate areas for reaction assembly and product analysis. Use primers spanning an exon-exon boundary. Non-specific PCR products can be eliminated by optimizing PCR reactions. This involves the following: (1) check your primers with a computer program, (2) increase annealing temperatures in 1°C increments, and (3) lower primer concentration to 75 nm.