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='''E. coli nucleus labeling using SYBR Green I and YOYO-1'''= ==Objective== *To confirm the bacteria staining with revised procedures **Using FITC filter, 20X objective, No cover slip *The stained bacteria will be used for microwell application ==Material and Samples== *Samples: Diluted E. coli, 10^6 CFU/uL (stored in PBS, 4C) *YOYO®-1 iodide (491/509) - 1 mM solution in DMSO *Diluted SYBR Green I (diluted in H2O, stored in 4C) ==Procedures== *Drop 1 uL of E. coli on the glass slide *Mix the E. coli with either 10 uL 2X SYBR Green I or 2 uL of YOYO-1 dye, gently pipetting for mixing *Leave the slide in dark box (slide storage box)for incubation 15 min in room temp ==Results== [[Image:SNAP-151939-0309-sybr-20x-B&W.jpg|SNAP-151939-0309-sybr-20x-B&W|600px]] Upper: SYBR Green I, 20X magnification, B&W [[Image:SNAP-153406-0004-sybr-20x-fitc.jpg|SNAP-153406-0004-sybr-20x-fitc|600px]] Middle: SYBR Green I, 20X magnification, FITC filter - Linear mode [[Image:SNAP-153514-0005-sybr-20x-fitc.jpg|SNAP-153514-0005-sybr-20x-fitc|600px]] Lower: SYBR Green I, 20X magnification, FITC filter - Best fit mode *The YOYO-1 pictures were not available due to overexposure problem -> YOYO-1 dye needs to be diluted before use. ==Discussion== *Both SYBR green dye and YOYO-1 dye can penetrate the bacteria and label on dsDNA without fixing bacteria *Both SYBR green dye and YOYO-1 dye can been seen under FITC filter (filter designed for FITC dye) *The SYBR green dye has fast photobleaching characteristics. Don't exposure to filtered light longer than 30 sec. The difference of emission reduced a lot after 30 sec. *After the excitation, the emission of fluorescent can last for a while even we turn off the exciataion. The colorful image still can be seen under the lamp light (white light). ==Suggestion== *Dilute the YOYO-1 dye before using -> find out best staining concentration. *For the real application on microwell array, it needs to washout the unlabeled dye background before loading on the array. *Ziess dye set list[https://www.micro-shop.zeiss.com/us/us_en/spektral.php?f=fa] *Ziess filter set list[https://www.micro-shop.zeiss.com/us/us_en/spektral.php?f=fi]
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