Comparison of Staining Dyes: Difference between revisions

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==Discussion==
==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.
(4/16/10)
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.
To help reassess the sample, we performed a follow-up experiment.
(4/22/10)
Because of SYBR Green's rather fast photo-degradation, we have decided to try and standardize our samples with 2X Dapi staining. The issue with Dapi is that it can produce saturated images, so the concentration must be carefully considered.


==Follow-up==
==Follow-up==
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===Discussion===
===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.

Latest revision as of 00:55, 28 April 2010

4/16/10[edit]

Purpose[edit]

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

Methods[edit]

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[edit]

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[edit]

(4/16/10) 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.

(4/22/10)

Because of SYBR Green's rather fast photo-degradation, we have decided to try and standardize our samples with 2X Dapi staining. The issue with Dapi is that it can produce saturated images, so the concentration must be carefully considered.

Follow-up[edit]

Methods[edit]

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[edit]

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[edit]

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.