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From 2010.igem.org

Module I, Group 2c

To solve the problem of severe dimerization of primers, we tried to adjust PCR system and use different concentration of upstream and downstream primers. As the concentration of PCR product is too low to be digested and purification, we attempted to use PCR product got yesterday as the template for PCR today.

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PCR system (pyrobest):

 H2O	                       17.3ul
 5×buffer	               2.5μl
 dNTP	                       2.5μl
 primer1	               1μl
 primer2	               0.5μl
 template	               1μl
 FastPFU	               0.2μl
 Total	                       20μl

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Result: to my disappoint, after gel purification,target fragments disappeared! What's worse, no PCR product of Chlr is seen in the gel photograph actually.