Can anyone suggest a fluorescent DNA-binding dye that gives the highest signal to background ratio in comet assay. Also can anyone suggest a good software that is suitable for yeast comets' analysis.
I agree with Johannes, because ethidium bromide is a unsafe staining. You can obtain SYBR Green easily and you can prepare a diluited stock solution in TE (ph 7.5) which is stable for several weeks when stored at 4 ° C in the dark. SYBR Green’s maximum excitation and emission are respectively 494 nm and 521nm. A fluorescein filter is adequate. The samples fluorescence is usually stable but you can use an antifade solution if fading of samples occurs.
There are several image analysis systems that are suitable for quantitation of Comet Assay data, e.g. 'Comet Assay IV' software but it's a pay-software. In my opinion you can also score your comets by using a free software as Casp: it works very well.
At least 50-75 randomly selected nucleoids should be analyzed per sample.
If you still have any questions, please don't hesitate to contact me
Good luck, Barbara
I´ve done the staining with SYBR Green and the image analysis with 'Comet Assay IV' software (Perspective Instruments, UK)
I agree with Johannes, because ethidium bromide is a unsafe staining. You can obtain SYBR Green easily and you can prepare a diluited stock solution in TE (ph 7.5) which is stable for several weeks when stored at 4 ° C in the dark. SYBR Green’s maximum excitation and emission are respectively 494 nm and 521nm. A fluorescein filter is adequate. The samples fluorescence is usually stable but you can use an antifade solution if fading of samples occurs.
There are several image analysis systems that are suitable for quantitation of Comet Assay data, e.g. 'Comet Assay IV' software but it's a pay-software. In my opinion you can also score your comets by using a free software as Casp: it works very well.
At least 50-75 randomly selected nucleoids should be analyzed per sample.
If you still have any questions, please don't hesitate to contact me
Good luck, Barbara
check this pubblication:
Photochem Photobiol Sci. 2007 Feb;6(2):181-9. Epub 2007 Jan 2.
Rufloxacin-induced photosensitization in yeast.
Catalfo A, Calandra ML, Renis M, Serrentino ME, De Guidi G.
If you want I'll be able to send you a pdf
Thank you very much, Barbara for very detailed answers. I have obtained this publication. It has nice protocol with references.
I've been using GelRed in the yeast comet assay. GelRed is a sensitive dye and it only bleaches very slowly (much slower than SYBR Green, from my experience).
SYBR green will be better choice.
Below link provides the information that you need.
http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery/Image-Detail.alternateID.g000895.html
we've used hoechst 33342 and CometScore software. also sybr green is suitable for this assay.
I used SYBR Green 1 for my comet assay experiments and the analysis by Tritek CometScore Freeware v1.5 image analysis software.
We are using EtBr because of it aivailebility and because it is giving very good results (signal-to noise ratio) and does not fade. However, it need to be handled with more care than other stains (like SybrGreen). However, also SybrGreen is an DNA intercalator...
Regarding the software: I rely on Perceptive Instruments Comet Assay IV. It is expensive but give good results in respect to quantitation. Data can be exported and analyzed using statistic tools. Other programs (free: ImageJ plugin, CometScore from TriTek or to be paid: Komet 5.5 Andor Scientific) gave less reliable results.
Kind regards and good luck,
Kristian
SYBR Gold works very well and doesn't fade like SYBR Green. Our lab also uses Comet Assay IV software from Perspective Instruments, UK.
SYBR Gold stain is >10-fold more sensitive than ethidium bromide for detecting DNA and RNA in denaturing urea, glyoxal, and formaldehyde gels, even with 300 nm transillumination. SYBR Gold stain has also been shown to be much more sensitive than SYBR Green II stain for detecting single strand conformation polymorphism (SSCP) products. Furthermore, it is much more sensitive than silver-staining for the detection of double-stranded DNA in native polyacrylamide gels.
Tuma RS, Beaudet MP, Jin X, Jones LJ, Cheung CY, Yue S, Singer VL. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Anal Biochem (1999) 268:278-288.
I've been using EtBr for many years but NP Singh thinks that YOYO-1 is probably the most appropriate DNA dye (Int J Radiat Bio, 66:23-28, 1994) . However, it is highly probable that this molecule is highly genotoxic!
I was thinking in using DAPI as well.
Any suggestions about EtBr/SYBR concentrations to use?
I think, silver staining is a Safest rather than another staining methods .
The following reasons:
-high quality images
-fast staining rather than silver-nitrate staining
-need a little material for staining
but this method is very toxic and not possible prepare archive for slides.
Dear Dmitry S Karpov,
you can try to freely download the 30day trial software using this link
cometassay.com
For the stain i think ethidium bromide, but it is carcinogenic and you have to use it under the hood with many precautions.
I have used Propidium iodide (PI) and it give good result over ETBR.
I have always had excellent comet assay results with Syber Gold. Etbr is highly carcinogenic and requires great care while handling.
What should be the fluorescent filter if I use ethidium bromide in the comet assay?
Please let me know?
I would suggest to use 590 nm filter for ethidium bromide. Details are here: http://archive.iorodeo.com/content/comparison-emission-filters-imaging-dna-gels-smartphone
A popular nuclear and chromosome counterstain, DAPI (4′,6-diamidino-2-phenylindole) emits blue fluorescence upon binding to AT regions of DNA.
I've always used 1x syber gold 1ul in 1000ul neutralization buffer and this works just fine. This amount can stain up to 20 slides (50ul each).
Can you please tell me the concentration of Propidium Iodide Ritika Rajpoot
5 to 10 micrograms of PI per mL, capture 10X fields as fast as you can
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