Hello Lourdes, I am not familiar with LR clonase or the protocol. From what I can glean, I assume you are linearizing the insert and vector with a restriction enzyme? If so, there are two potential issues. One, the digested products may just be recircularizing following transformation. Two, digestion may be incomplete so the original vectors, that are likely supercoiled, will be preferentially taken up by the cells. I would recommend PCR amplifying the vector and insert and using low concentrations of each. Following PCR, use DpnI to selectively digest the vectors leaving only the amplicons. Following PCR cleanup, you can then proceed with the LR clonase. Good luck!

Hi Jamison Drew Law ! Thanks for your answer

Let me check if I understand... Are you suggesting that I recombine the target vector with the PCR-amplified gene fragment? I never tried that before

Lourdes, I am recommending that you PCR amplify the vector AND inserts to linearize, DpnI digest to get rid of the original templates (by virtue of them being methylated), PCR cleanup, and then proceed with the LR clonase reaction. Of course check each PCR product before and after PCR cleanup. Good luck!