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Different dyes for gel electrophoresis


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9 replies to this topic

#1 jiajia1987

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Posted 08 February 2009 - 05:47 PM

Hello people,

I have been using Gelstar for gel electrophoresis and I realized that the gel results always appear different from when I use SybrGreen for gel electrophoresis. Just what are the differences between Gelstart, SybrGreen and ethidium bromide and do they give different results on gel?

Many thanks!!

#2 chrisbelle

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Posted 09 February 2009 - 10:01 PM

Hello people,

I have been using Gelstar for gel electrophoresis and I realized that the gel results always appear different from when I use SybrGreen for gel electrophoresis. Just what are the differences between Gelstart, SybrGreen and ethidium bromide and do they give different results on gel?

Many thanks!!


What do you mean by different? All the above are DNA intercalating dyes, meaning that they stick between two DNA strands. Ethidium bromide is the standard dye used, and may be carcinogenic, that's why they come out with other dyes like SYBR and gelstar. But gelstar can stick to ssDNA and RNA as well. I would say that sybr and gelstar would be more sensitive, but i need to know what you mean by gel results appear different...

Chris
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#3 jiajia1987

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Posted 09 February 2009 - 10:59 PM

Hello people,

I have been using Gelstar for gel electrophoresis and I realized that the gel results always appear different from when I use SybrGreen for gel electrophoresis. Just what are the differences between Gelstart, SybrGreen and ethidium bromide and do they give different results on gel?

Many thanks!!


What do you mean by different? All the above are DNA intercalating dyes, meaning that they stick between two DNA strands. Ethidium bromide is the standard dye used, and may be carcinogenic, that's why they come out with other dyes like SYBR and gelstar. But gelstar can stick to ssDNA and RNA as well. I would say that sybr and gelstar would be more sensitive, but i need to know what you mean by gel results appear different...

Chris


Hello Chris,

I am sorry I didn't make things clear enough.

One thing I noticed was that the gel band intensity upon illumination is ascending in this order: gelstar, sybrgreen and ethidium bromide. for the same volume of DNA and loading dye loaded, gelstar gives the brightest band at the same voltage and time used for running. And, the band can be so bright that it appears to be a smear. What appears to be a smear for gelstar actually provides a clearer and cleaner band in the case of sybrgreen. Sometimes, the bright band for gelstar is actually two bands in sybrgreen. I have heard that gelstar is much more sensitive and the amount of DNA loaded and loading dye added can be reduced by 5- to 10-fold.

I have also realized that gelstar is great for gel purification. Given the thick and bright band it provides upon illumination, it makes life easier when it comes to cutting of bands for gel purification. Sybrgreen, on the other band, shows up as a fainter band and it takes a while for the eye to get used to seeing the band. In the case of ethidium bromide, the band gets even fainter!

#4 chrisbelle

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Posted 11 February 2009 - 01:20 AM

Hi jiajia,

That's good! I mean so scientists actually made something that made our lives easier! hahahaha
kidding. then I don't see your problem. in fact thanks for sharing with us the info on the 3 dyes. Yes highe intensity is to be expected with gelstar, and if you expect your band to be thick then use less DNA and dye when you run your gel.

Chris
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#5 jiajia1987

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Posted 11 February 2009 - 09:32 PM

Hi jiajia,

That's good! I mean so scientists actually made something that made our lives easier! hahahaha
kidding. then I don't see your problem. in fact thanks for sharing with us the info on the 3 dyes. Yes highe intensity is to be expected with gelstar, and if you expect your band to be thick then use less DNA and dye when you run your gel.

Chris


Hi Chris,

It was a 'problem' in the sense that I was always wondering why my inserts or plasmids were of a higher mass than expected on the gel when I used gelstar until I started using other dyes such as Sybrgreen. I thought that I had the wrong insert or plasmids. This went on for a month! I was just wondering if other people have noticed these differences when they used different dyes for gel electrophoresis. I thought it would be great to have some opinions just for verification's sake. <_<

#6 jiajia1987

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Posted 13 February 2009 - 02:32 AM

I am sorry to add on this.


I seem to always see slight variations in the migration of different lanes in the same gel for SybrGreen. Bear in mind that the different lanes are the same samples. Did anyone encounter this? Does anyone know how SybrGreen can affect DNA migration?

Edited by jiajia1987, 13 February 2009 - 02:33 AM.


#7 hanming86

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Posted 15 February 2009 - 08:52 AM

I don't think you should be wondering much about all these matters. The fact that it works should get you moving on with your project well unless your project is comparing these three different DNA staining method.

Anyway, binding of SYBR to DNA might have just changed their overall charge thus causing the difference in migration pattern.

I normally stain after electrophoresis. time consuming yes but it's less messy and probably safer overall.
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#8 jiajia1987

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Posted 15 February 2009 - 07:07 PM

I don't think you should be wondering much about all these matters. The fact that it works should get you moving on with your project well unless your project is comparing these three different DNA staining method.

Anyway, binding of SYBR to DNA might have just changed their overall charge thus causing the difference in migration pattern.

I normally stain after electrophoresis. time consuming yes but it's less messy and probably safer overall.


Dear hanming86,

I am wondering about all these matters because I am doing a final year project, which takes up to 3 to 4 months. This is a relatively short period and it is insufficient for me to come up with major results within this time frame. Instead, what we are encouraged to do during our final year project is to experience for ourselves hands-on work in a company and to learn to troubleshoot things. It is critical for us to take note of different things in the lab and as such, I am wondering about the differences in the dyes used for gel electrophoresis because all these three dyes produced slightly different results.

I am currently trying to find out the effect of SybrGreen on DNA migration because my supervisor said SybrGreen has a big effect on that.

Cheers,
Jiajia1987

#9 molgen

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Posted 16 February 2009 - 03:05 AM

I seem to always see slight variations in the migration of different lanes in the same gel for SybrGreen. Bear in mind that the different lanes are the same samples. Did anyone encounter this? Does anyone know how SybrGreen can affect DNA migration?


Do you mean by this that you get a smiley?
That is, dos the middle of the gel runs farther than the sides?
If so, the reason is differences in the electric field. There is more resistance in the side then in the middle.

#10 jiajia1987

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Posted 18 February 2009 - 05:19 AM

I seem to always see slight variations in the migration of different lanes in the same gel for SybrGreen. Bear in mind that the different lanes are the same samples. Did anyone encounter this? Does anyone know how SybrGreen can affect DNA migration?


Do you mean by this that you get a smiley?
That is, dos the middle of the gel runs farther than the sides?
If so, the reason is differences in the electric field. There is more resistance in the side then in the middle.


Hi molgen,

I do not mean smiley, but thanks a lot for telling me about what you did.

What I mean is that even though the products are of the same size, eg, 1.5kb, and I run them in different lanes, the bands from each lane will not all be on the same level. Some will be higher up, some will be lower.

I hope you get what I mean.




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