Day 1Thaw cells, grow 24-48 h. Day 2Restreak, grow 18-24 h. Day 3Early in the day: Streak cells in a thin layer onto BHI + 2% yeast (BHI+Y) (yeast is optional but is helpful). Grow 6 h.Pre-warm 1-2 BHI+Y plates.Harvest 6-h plate of cells in 2 mL PBS, wash 1x in PBS, resuspend in 250 uL PBS.Spot entirety of cell suspension onto pre-warmed BHI+Y plates by filling a pipette tip and spotting discrete ~20-uL drops across the plate(s).Pipette plasmid DNA (20 ng/uL in water) atop each spot (~10-20 uL drops). Let sit to dry a few mins. Incubate O/N (right-side-up). Day 4Pre-warm antibiotic-containing BHI plates (will need ~6)Streak growth from cell+DNA spots onto BHI + antibiotic plates in a thin layer (streak out as much of the growth as you can - use several plates). Incubate 2-5 days, checking each day for colonies. Day 6 (or whenever colonies are observed)Patch colonies onto a new selective plate and grow overnight. Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Day 2Restreak, grow 18-24 h. Day 3Early in the day: Streak cells in a thin layer onto BHI + 2% yeast (BHI+Y) (yeast is optional but is helpful). Grow 6 h.Pre-warm 1-2 BHI+Y plates.Harvest 6-h plate of cells in 2 mL PBS, wash 1x in PBS, resuspend in 250 uL PBS.Spot entirety of cell suspension onto pre-warmed BHI+Y plates by filling a pipette tip and spotting discrete ~20-uL drops across the plate(s).Pipette plasmid DNA (20 ng/uL in water) atop each spot (~10-20 uL drops). Let sit to dry a few mins. Incubate O/N (right-side-up). Day 4Pre-warm antibiotic-containing BHI plates (will need ~6)Streak growth from cell+DNA spots onto BHI + antibiotic plates in a thin layer (streak out as much of the growth as you can - use several plates). Incubate 2-5 days, checking each day for colonies. Day 6 (or whenever colonies are observed)Patch colonies onto a new selective plate and grow overnight. Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Day 3Early in the day: Streak cells in a thin layer onto BHI + 2% yeast (BHI+Y) (yeast is optional but is helpful). Grow 6 h.Pre-warm 1-2 BHI+Y plates.Harvest 6-h plate of cells in 2 mL PBS, wash 1x in PBS, resuspend in 250 uL PBS.Spot entirety of cell suspension onto pre-warmed BHI+Y plates by filling a pipette tip and spotting discrete ~20-uL drops across the plate(s).Pipette plasmid DNA (20 ng/uL in water) atop each spot (~10-20 uL drops). Let sit to dry a few mins. Incubate O/N (right-side-up). Day 4Pre-warm antibiotic-containing BHI plates (will need ~6)Streak growth from cell+DNA spots onto BHI + antibiotic plates in a thin layer (streak out as much of the growth as you can - use several plates). Incubate 2-5 days, checking each day for colonies. Day 6 (or whenever colonies are observed)Patch colonies onto a new selective plate and grow overnight. Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Day 4Pre-warm antibiotic-containing BHI plates (will need ~6)Streak growth from cell+DNA spots onto BHI + antibiotic plates in a thin layer (streak out as much of the growth as you can - use several plates). Incubate 2-5 days, checking each day for colonies. Day 6 (or whenever colonies are observed)Patch colonies onto a new selective plate and grow overnight. Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Day 6 (or whenever colonies are observed)Patch colonies onto a new selective plate and grow overnight. Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Day 7Perform colony PCR using gene-specific or vector-specific primers to check for insert DNA.Streak out full plates of growth from at least 3 different positive colonies in order to make frozen stocks.Tip: It can be easier to transform 81-176 than 11168, so if you’re having trouble getting DNA into 11168, try putting pDNA into 81-176 first, extracting pDNA from those cells, and transforming that into 11168.