( high GC content that do ...)QIAGEN Multiplex PCR Handbook

high GC content that do not amplify under these conditions should be combined in a separate multiplex PCR assay using 1x Q-Solution. If detecting multiplex PCR products under non-denaturing conditions (e.g., on agarose gels or native polyacrylamide gels): Some products are smeared, or additional products are observed 1. Too many PCR cycles Too many PCR cycles may increase nonspecific background. Determine the optimal number of cycles by decreasing the number of PCR cycles in increments of 3 cycles. 2. Too much starting template Check the concentration of the starting template DNA. Repeat the multiplex PCR using less DNA (i.e., <= 1 g per 50 l reaction). 3. No final extension step, or final
extension step was not optimal Ensure that the final extension step was performed as described in Table 3, Table 5, and Table 7. When detecting multiplex PCR products under native conditions, a final extension step of 15 min at 68C for multiplex systems with more than 10 PCR products, or for PCR products longer than 1.5 kb may improve results. For microsatellite analysis, a final extension step of 30 min at 60C should be used. 4. Incomplete renaturation of PCR
products due to either low GC
content or long length of
PCR products Use a final extension step of 15 min at 68C. We recommend this for multiplex systems with more than 10 PCR products, or for PCR products l
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