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==Procedures== *Sample preparation: #Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes #Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL PBS. #Transfer into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. Resuspend in 1mL PBS. *Prepare 100uL sample for staining. #Prepare six 1.5-mL tubes. Transfer 100uL cell suspension into each of tubes. #Fresh-Ecoli x3 (SYBR-gold, YoYO, DAPI), Dead-Ecoli x3 (SYBR-gold, YoYO, DAPI) ## Dead E.coli were from the same source as on 4-21-'11's test. The E.coli cells cultured on 3-10-'10, stored in 4C. #Pellet cells at 8000rpm, 5min, RT. Remove supernatant, resuspend in 0.5mL PBS. *Prepare following DNA staining dye SYBR-GOLD(10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X YOYO-1 (10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X DAPI (pre-diluted 5mg/mL) -> 1/5 dilution with H2O -> 1mg/mL #Add 2uL of diluted dye into sample suspension (0.5mL). Repeat pipetting slowly ~20X to mix the dye with sample. #Incubate in the Eppendorf rocking incubator at 25C, 400~500rpm for 15min.
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