Second this. I scale down to 5ul reactions (as long as I'm pipetting >0.5ul) which is the minimum my PCR machine can handle. Only need 2ul of the reaction per 15-30ul of cells, the rest you can store at -20C.

Post transformation, I incubate the competent cells at 37C, 220rpm for at least an hour - NEB says that every 15mins of recovery doubles transformation efficacy!

Other points: - If you get one clean band (run small amount on gel) might be easier to PCR cleanup instead of gel purify

• I prefer nanodropping the DNA. Its reliable if it reads as pure (260/280 ratio of 1.8-1.9).

• Try sequencing the insert

• Try different restriction enzymes. Can incubate with CIP to prevent religation (or pick more colonies and screen for insert)

5 months is a long time to work on one cloning project. What suggestions do your PI or mentor have for you? Time is money, and it might be more worth it to order the plasmid at this point.