Helllo All 👋!
I am working trying to assemble dsDNA fragments (for further golden gate domestication) via polymerase cycle assembly (PCA). After designing the required oligonucleotides with overlap regions. (Image of the design provided below). Using
NZYProof Green mastermix x2 25 uL Inner Oligonucleotides 2 uL of 1 uM stock Outer Oligonucleotides 2 uL of 100 uM stock
Initial denaturation 95 ºC 2 min Denaturation 95 ºC 30 sec Annealing 62 ºC 30 sec Extension 72 ºC 30 sec Repeated by 25 cycles Final extension 72 ºC 2 min
After performing the following protocol (based on This one) with a high fidelity polymerase mastermix, the EF gel of PCR products only shows a single "band" corresponding with the oligonucleotides introduced in the reaction. (Ignore the blurred EF gel, it was imaged quite a long time after running the gel).
Any idea of what could actually be going wrong? Should not I observe at least some faint higher molecular weight amplicons?
Thanks in advance for your answers!
UPDATE: Regarding the length of overlap regions, the design considers different overlap sequences length in order to achieve roughly the same Tm for all the overlaping regions. Tm of every overlap has been calculated using the online NEB Tm Calculator tool, and all of them are in the range of 61-63 °C (Considering a Q5 mastermix, which is the closest HiFi polymerase to the one we are currently using )
UPDATE2: After trying the PCA procedure with a "touchdown" approach (reducing annealing temperature gradually after 3 succesive cycles, most of the assemblies has started to show distinctive bands of the desired lenght in the EF Gel. Annealing is a crucial parameter for PCA.