Additional cons"when you do a large scale 2-hybrid screen CR amplification gives mutations -generally dont sequence everything to confirm Cloning transformation inefficiencies If baits are pooled slow-growing cells will lose to faster ones, giving false negatives All VS all assay contains many implausible interactions - proteins that arent Co-localized or expressed at the same time Can only sequence a small fraction of the positive clones High-throughput Y2H screens miss as many as 90% of Y2H interactions observed in focused, small-scale studiesAdditional “cons” when you do a large scale 2-hybrid screen PCR amplification gives mutations - generally don’t sequence everything to confirm! Cloning & transformation inefficiencies If baits are pooled, slow-growing cells will lose to faster ones, giving false negatives. All vs. all assay contains many implausible interactions - proteins that aren’t co-localized or expressed at the same time. Can only sequence a small fraction of the positive clones. High-throughput Y2H screens miss as many as 90% of Y2H interactions observed in focused, small-scale studies!