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In designing primers for PCR, the following steps/rules were tested and proven to be useful:
Fig. 7. Multiplex PCR using primers 18-24 bp long
When PCR reaction Eight individual loci are amplified with similar intensities when the primer pairs are used separately. When equimolar amount of these primers are mixed together for a multiplex reaction (Mix K), some of the products are much weaker (#1, #2, #5, #6) than other. In this case, primers had "usual" length, between 18-24bp.
(primers used in this case amplify polymorphic loci, explaining the "double" or "triple" bands as seen on a regular agarose gel)
Fig. 8. Multiplex PCR using primers 30-35 bp long
Compared to the figure above, in this case the primers used for multiplexing were longer than 30 bp (up to 37 bp). Equimolar amounts of primer were used and all loci were amplified with comparable intensities in each reaction.