Sam:LabNotes/Microbiome/2009-3-9

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Revision as of 00:17, 10 March 2009 by >Sam Chiang (→‎Suggestion)
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E. coli 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

  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

  • Both SYBR green dye and YOYO-1 dye can penetrate the bacteria and label on dsDNA without fixing bacteria
  • 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[1]
  • Ziess filter set list[2]