( refer to Appendix. ...)QIAGEN Multiplex PCR Handbook

refer to Appendix. 4. Insufficient number of cycles Increase number of PCR cycles. Refer to Appendix, for guidelines. 5. PCR cycling conditions not optimal Check that the correct cycling conditions were used (see Table 3, Table 5, and Table 7). Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing
temperature. 6. Primers degraded or of low quality Check functionality and specificity of primer pairs in single PCR reactions. Ensure that primers of sufficiently high quality were used. Check for possible degradation of the primers on a denaturing polyacrylamide gel. If necessary, make new dilutions of primer mix from primer stock solutions and store at 20C in small aliquots. Avoid repeated freeze/thaw cycles of the primer mix. 7. Annealing temperature too high Follow the recommendations given in Table 10 to determine the appropriate annealing temperature for your primers. Decrease annealing temperature in increments of 3C. Ensure that an annealing time of 90 s was used. If possible, perform a gradient PCR to determine the optimal annealing temperature. 8. GC-rich template or template
with a high degree of
secondary structure Using the same cycling conditions, repeat the multiplex PCR using 0.5x Q-Solution. Templates with a very high GC content that do not amplify under these conditions should
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