Sam:LabNotes/Microbiome/2009-3-9: Difference between revisions
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>Sam Chiang (Replacing page with '* '''E. coli nucleus labeling using SYBR Green I and YOYO-1'''') |
>Sam Chiang |
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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] |
Latest revision as of 21:42, 16 March 2009
E. coli nucleus labeling using SYBR Green I and YOYO-1[edit]
Objective[edit]
- 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[edit]
- 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[edit]
- 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[edit]
SNAP-151939-0309-sybr-20x-B&W Upper: SYBR Green I, 20X magnification, B&W SNAP-153406-0004-sybr-20x-fitc Middle: SYBR Green I, 20X magnification, FITC filter - Linear mode SNAP-153514-0005-sybr-20x-fitc 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[edit]
- 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[edit]
- 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[1]
- Ziess filter set list[2]