1. What should I do if no product is obtained with unique sequence primers? This result can occur for several reasons. The simplest explanation is that one of the reagents was omitted or is "old." Aliquots of PCR reagents, such as 10X buffer and dNTPs should be stored at -20oC to prevent their degradation. This possibility can be controlled for by including a positive control, such as the primers included with the kit. It is also possible that the mRNA source you have chosen express the target gene at very low levels. We recommend that you try several of the Multiple ChoiceTM cDNAs in your initial screening. 2. What should I do if no product is obtained with degenerate primers? It is important to determine whether the failure to obtain a PCR product is due to any of the reasons listed for unique sequence primers. Once you have eliminated all of the possibilities listed above, there are several other likely causes for the lack of product with the degenerate primers. a. The primers may contain too many mismatches. You can try reducing the annealing temperature or you can construct new primers. b. If you are trying to amplify the 5' end of a very long mRNA, we recommend that you use cDNA which was primed with random hexamers rather than this cDNA which is oligo(dT)-primed. 3. What should I do if too many PCR products are generated? Try increasing the stringency of the PCR reaction by either increasing the annealing temperature or reducing the primer concentrations. Additionally, tetramethylammonium chloride (TMAC), formamide and MgCl2 have been reported to increase the specificity of PCR reactions using unique sequences. Alternate primers can also be constructed or a "nested" PCR approach can be used.