Hello, I'm facing a conundrum and was hoping to get some advice.

We have 12 cryo tubes (all replicates of the same clone) of HEK293T cells preserved in our -80 that have supposedly had a plasmid cassette inserted into the AAVS1 safe harbor locus site (I can provide more info on this if needed). I say supposedly because we're trying to verify this using various means, including PCR.

One of the replicates is showing promise in our PCR screenings. It should match one of the 12 replicates in the freezer. The problem is, I don't know which one. The genomic DNA we've been doing PCR screening with was labeled with well numbers, while the preserved cells only have the cell count and the clone number (they're all clone 1). Obviously this was an oversight we're now paying for.

I'm discussing with my PI how to go about screening them via western blot, which would involve thawing them all out, taking a portion for whole cell extract and another portion to re-preserve.

The preserved tubes range in cell count from ~3 x 10^6 cells/mL to just 1.55 x 10^4 cells/mL. For the tubes with lower counts, can they still be thawed in a T25 or do I need to use something smaller?

I just don't see any other way to go about this other than thawing them all out, but if I'm missing something, I'm open to suggestions! Thanks!