Comparison of Staining Dyes

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Revision as of 00:02, 28 April 2010 by >Jbrubake (→‎Results)
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Purpose

To establish the best dye to use for any subsequent experiments in order to maintain consistency in results.

Methods

3 dyes were tested: Dapi, SYBR Green, and YOYO. Each dye was diluted to 2X concentration.

Sample tubes 1 and 2 were combined, filtered through a 5um filter, and used as a standardized sample.

For each dye:

- Use a clean 0.2mL tube
- Add 7uL sample + 3uL 2X dye
- Incubate for 10 minutes in the dark
- Add 10uL water
- Vortex and spin down

Each sample was plated and imaged under a fluorescence microscope using 10X magnification.

Results

File:Dapi 10X Comparison.jpg

Image 1: 2X Dapi

File:SYBR Green 10X Comparison.jpg

Image 2: 2X SYBR Green

File:YOYO 10X Comparison.jpg

Image 3: 2X YOYO

Discussion

Even though the images appeared to be dirty, SYBR Green showed the clearest image, so we decided to stick with SYBR Green for the time being. However, we were unsure whether or not glycerol in the sample affected the image quality.

To help reassess the sample, we performed a follow-up experiment.

Follow-up

Methods

Two slides were extensively cleaned: washed down with isopropanol, wiped with a Kimwipe, washed again with isopropanol, and air-dried in the fume hood.

To one slide a 20% glycerol mixture was added, with 2X SYBR Green as the dye. To the other slide, 7uL sample + 3uL 2X SYBR Green was added.

Results

File:20%Glycerol 10X.jpg

Image 1: 20% Glycerol strained with 2X SYBR Green

File:Sample cleaned 10X.jpg

Image 2: Sample stained with SYBR Green

Discussion