Daniel:Notebook/ComboLock/2016-10-31
Jump to navigation
Jump to search
Circularization Optimization Testing
There are several potential difficulties going on right now, but I think the circularization step is the weakest in the protocol. I'm going to test several different methods of circularization:
- T4 polymerase vs Klentaq; according to NEB Klentaq may degrade an encountered downstream strand, which would be a problem potentially. T4 and T7 do not have strand displacement, which will be helpful for circularization
- Time; from what I understand less time is fine and it will be a helpful thing sometimes to not do an overnight step
- RCA; the original idea called for RCA on the final circularized product and not just PCR. Doing some RCA first would greatly increase the amount of product available for PCR and would likely therefore reduce the amount of off-target product such as primer dimers.