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===Discussion=== * All 5 polymerases worked in Ampligase buffer but they vary significantly. I plan to repeat the experiment using duplicates (but vary input: 100ng and 200ng) to confirm that these differences are real. * I plan to move capture reaction onto ice when adding ExoI/III. I believe that when the reaction cools to 37C on the thermocycler, there may be an opportunity for Ampligase to ligase unused probes together. After discussing with others in the lab, we wonder if Ampligase may still have activity after incubation for long periods at ~55C, if it does, then adding ExoI/III on ice may be crucial to reducing background levels. * I will continue performing capture PCR with Phusion Flash as this mix seems to reduce the background signals between 200-400bp. * Since the concentration of HemoKlentaq is not known, we usually assume it to be 10U/uL and use 5 U (0.5 uL). For this experiment, I assumed the concentration to be 5 U/uL, and used 3 U (0.6 uL). The amount of HemoKlentag that I used is very close to the usual amount.
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