In the process of developing dLUTE SEQ CF we have discovered a simple change to the thermocycling conditions greatly improves the robustness of the dLUTE reaction.
This change is the addition of a 3 minute 75˚C preincubation step before the main cycling steps. This step allows the dLUTE phases to separate before the DNA polymerase in the sequencing mixture is exposed to denaturing temperatures. This appears to prevent the polymerase from inactivating when exposed to the large surface area of the inert phase in its intact form.
I will give this a try.
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