Comparison of Staining Dyes
4/16/10
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
Image 1: 2X Dapi
File:SYBR Green 10X Comparison.jpg
Image 2: 2X SYBR Green
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
Image 1: 20% Glycerol strained with 2X SYBR Green
Image 2: Sample stained with SYBR Green
Discussion
Unexpectedly, both the glycerol control and the samples had masses show up under the microscope. This is odd because the slides were thoroughly cleaned and the glycerol did not have chromosomes added to it. This suggests that glycerol does in fact bind to the SYBR Green dye.