My lab is working on a simple library prep and are running into an issue with our amplification PCR, our samples do not successfully amplify. After preforming the reaction (using 2X Phusion HF Polymerase, water, and our primers) for 12 cycles, the samples very low concentrations according to our qubit. In addition, when run on a gel we see nothing (including no primer dimers).
However when we use this product and run a standard QC PCR, the DNA amplifies successfully and can be seen on a gel.
For trouble shooting we have tried running the reaction with KAPA HiFi HotStrt ReadyMix and running the reaction for both fewer cycles (in case it was being overloaded) and for a greater number of cycles.
Has anyone run into this problem before or have other suggestions for trouble shooting?
However when we use this product and run a standard QC PCR, the DNA amplifies successfully and can be seen on a gel.
For trouble shooting we have tried running the reaction with KAPA HiFi HotStrt ReadyMix and running the reaction for both fewer cycles (in case it was being overloaded) and for a greater number of cycles.
Has anyone run into this problem before or have other suggestions for trouble shooting?