I´m planning to Synchronize HEK293T cells at the boundary of G1/S phase - using Double-Thymidine Block - before performing Transfection (using Lipofectamine). The aim is to transfect plasmids coding for Cas9, gRNA and Donor - respectively - and to see if it is possible to observe increased HDR events compared to the un-synchronized. Although there is plenty of protocols coming from the paper, it is not really clear the time point of the Transfection. Briefly: the syncronisation should be done by pre-seeding the cells at low concentration 16/17h before, then add a final concentration of 2 mM of thymidine to the medium and incubate for 17/18h, then release the cells by washing the cells and adding medium without thymidine and cultivate for 9h and then again adding the thymidine 2mM and 17h incubation. Finally, release the cells by switching to medium without thymidine. My question is at which point should I peform the transfection? Since it is gonna be plasmids, it will take some time for them to be expressed, in particular for the Cas9 to be translated. So I was wondering which would be the best time point to transfect the cells so that I´m sure that the block is still in place when the Cas9 starts cleaving the DNA.

I was thinking about performing the transfection during the second thymidine administration(maybe in the second half i.e. 8h later), to ensure that by the time I release the cells and let them progress through the S phase the Cas9 is ready to cleave.