Talk:Agarose gel electrophoresis/Archive 1

Kudos & Photo opp

Nice job on the page. I especially like the diagram. Would it be helpful to place an actual (black and white) photograph of an agarose gel in the article? It could probably fit next to the diagram. I'm not working with agarose gels right now, but I can get a photo rather easily. (if others think it would be helpful) AdamRetchless 04:49, 25 Mar 2004 (UTC)

Often articles use direct black and white photos of an agarose gel as figures as well, so yes it would be useful. I'd *really* love to see a color picture taken at an oblique angle sometime though. It's a nice thing to look at. :-) Kim Bruning 23:20, 24 Apr 2004 (UTC)

It would be useful if you could obtain a photo of a gel under room light and the same gel stained with ethidium bromide under UV illumination, the former showing the dye front and the latter showing the orange glowing bands; one including a glowing DNA ladder would be good. Also, if one could obtain pictures of an RNA gel (relatively common) that would be useful too, showing 28s and 18s RNAs which are typically used internal size markers for eukaryotic RNA separations ... would need to be total RNA then rather than poly-A or size fractionated or poly-ribosomal. Courtland 2005-01-31 USA 20:45 EST

Ethidium Bromide

Justification for the fact that I reverted the addition of latex gloves:

Although commonly used to protect against ethidium bromide, they are not the best choice. Nitrile gloves are better.

Here are links with different levels of support for this view:

• www.princeton.edu/~ehs/wastepaper/99-4.html => http://web.princeton.edu/sites/ehs/chemwaste/WastePaper/0302.htm Courtland
• http://www.mbl.edu/labs/Sogin/Pages/SoginLabGuide.html
• www2.fpm.wisc.edu/biosafety/Base/bioside_lines0403.htm => http://www2.fpm.wisc.edu/biosafety/bioside_lines/bioside_lines_04_2003.htm Courtland
• http://www1.qiagen.com/literature/qiagennews/0200/Practical%20Hints.pdf
• www.scripps.edu/services/ehs/SafetyGram/SG1999/SG1999B.htm => http://www.scripps.edu/researchservices/ehs/News/safetygram/sg1999/sg1999b.html Courtland

Delta G 05:22, 3 Apr 2004 (UTC)

Considering how commonly (and generously) used EtBr is, it's a shame that a typical MSD on this solution isn't more informative on this matter, only saying "appropriate gloves". See http://www.sefsc.noaa.gov/HTMLdocs/EthidiumBromide.htm If someone has seen an MSD that has better protective information, could you reference that here? Courtland 2005-01-31 USA ~19:50 EST

ethidium bromide fluoresces pink in the presence of DNA.

Surely EtBr fluoresces pink even in the absence of DNA? The reason DNA is visualised is because the EtBr interchelates between the bases and so is more concentrated where DNA is present.--Alun 18:22, 13 May 2005 (UTC)

Actually it doesn't, I checked and it looked green to me. In any case, the fluorescence yield increases when it is bound to DNA, and apparently the spectra changes.128.192.79.29 20:16, 20 February 2007 (UTC)

No mention of biomolecules other than double-stranded DNA (which is implied)

Gel electrophoresis is used for RNA too --nixie 05:40, 31 Jan 2005 (UTC)

Agreed. Agarose gel electrophoresis is also used for protein separation (which is mentioned in the introductory paragraph), particularly for zymography, and for oligonucleotide purification. Courtland 2005-01-31 USA ~20:40 EST

in other encyclopedias?

It doesn't look like Brittanica has an article specifically on this topic; did a quick search for 'agarose gel electrophoresis' at http://www.britannica.com/. Courtland 2005-01-31

Mistake?

As far as I know, Ethidium Bromide does not uncoil DNA.

It actually does, though probably "uncoil" is not the best term to use. When it intercalates into the strands of a plasmid, it changes the topology of the bases by relieving some of the negative supercoiling associated with it (and if you add enough, it will make the plasmid positively supercoiled). This can also be used to purify plasmids (since their taking up different amounts of EtBr changes their properties by differing degrees). But no, I don't believe it can actually pull the double helix apart. Arc de Ciel (talk) 03:24, 25 January 2010 (UTC)

Regarding migration of conformations...

The entry for Agarose Gel Electrophoresis states:

"The rate of migration is affected by a number of factors. The concentration of agarose is one that has been mentioned. The conformation of DNA is also a factor and is demonstrated by the three forms of a plasmid: superhelical, nicked, and linear. Each form runs differently, the superhelical the fastest and the linear form the slowest."

With regards to the statement about conformation, is the order of migration not (smallest to largest): superhelical (supercoiled), linear and then nicked?

Given plasmids of the same size, the supercoiled ones will move the furtherest, and the linearized ones will move the shortest. Nicked plasmids will end up somewhere in between, because they are partially coiled. —The preceding unsigned comment was added by 128.192.79.29 (talk) 20:25, 20 February 2007 (UTC).

As random phosphates are cleaved (or nicked) on both strands of the DNA, the DNA's superhelical state relaxes and completely unravels, but doesn't linearize. Therefore, it does not migrate as far as a linear DNA molecule (of the same size).

Also interesting to mention are those DNA molecules that migrate even slower than nicked DNA. Multimers of circular DNA, called catenanes, exist as dimers, trimers, etc., of linked circular DNA and typically migrate the slowest of all due to their weight.

-JTP 28 Oct. 2005, 00:35 AM EST

From the POV of someone who works in molecular biol, i agree, but cannot back up your comment on nicked DNA travelling slowest, then linear, then supercoiled having the fastest migration rate. Although the RF forms are always taught to students, noone i have spoken too has any good explanation for each band, and there is always a lot of hand waving... -JohnB 30mar2006

i had a problem with loading EtBr with the sample.. n after 45 min the fluresence wad seen on the other side of the well/ wad is the charge of EtBr

Procedure

Is there a reason that this belongs in an encyclopedia article? Seems more appropriate for Wikiversity.Nathanww 15:31, 20 July 2007 (UTC)

It seems to me that you can't really understand the article unless you can look at a simplified version of the procedure. —Preceding unsigned comment added by 128.104.41.112 (talk) 21:24, 21 October 2010 (UTC)

Gel picture

i doubt that the marker is a ibp marker, i assume it's a 1Kbp marker!!! — Preceding unsigned comment added by 194.95.224.172 (talk • contribs) 29 January 2008

Human waste?

Is agar also made from human waste?!

• No, it's produced from certain species of red algae. Take a look at our agar article for details. – ClockworkSoul 13:50, 1 April 2008 (UTC)

anyone heard of GelRed?

I removed this poorly cited and edited sentence since it sounded like an advertisement to me. If anyone can vouch for it, please feel free to replace with a citation: "GelRed, a nucleic acid stain developed by Biotium [1], is a safe alternative to ethidium bromide. The dye has almost identical absorption and emission spectra as EtBr, so no change in instrumental setting is need. GelRed is also nonmutagenic, noncytotoxic and environmental safe."

cheers, Mote (talk) 06:02, 2 June 2010 (UTC)

Merge with article on Gel Electrophoresis?

Another article exists on gel electrophoresis. The content overlaps considerably with this entry. I'm putting out there the suggestion to merge the two articles. 68.194.106.90 (talk) 19:52, 10 August 2010 (UTC)

Assessment comment

The comment(s) below were originally left at Talk:Agarose gel electrophoresis/Comments, and are posted here for posterity. Following several discussions in past years, these subpages are now deprecated. The comments may be irrelevant or outdated; if so, please feel free to remove this section.

Comment(s)

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The article has some has some good points but has the following correctable faults. I will try to address these. 1. Confusing use of 'strands' in the first section. No mention of RNA. Changed TransControl 23:24, 2 March 2007 (UTC) 2. Does not list how or why it is usually used. 3. Diverges into long sections on EtBr (indeed most of the current references are on this) when there are excellent resources on the web about agarose gel electrophoresis. Changed TransControl 23:24, 2 March 2007 (UTC) 4. Irrelevant material regarding 2D protein gels, should be elsewhere. Changed TransControl 23:24, 2 March 2007 (UTC) 5. Resolution limits are misleading, most experiments would separate fragments 100bp 20kbp at most. PFE is a separate technique requiring different specialised equipment, should be another article. PFE article now linked. 6. The details on the 'Preparation' and 'Procedure' are misleading, for example 'Boil solution' implies doing more than bringing it to the boil to dissolve the agarose. As most researchers who have boiled agarose by accident know, boiling for long will ruin your gel and is likely to boil over in the microwave. I think this detailed protocol is not really appropriate for an article, links to external sites with detailed protocols would suffice. 7. The gel photos are not well described or very informative. (replaced first gel picture Dr d12 00:22, 7 March 2007 (UTC)) Yes very good TransControl 09:35, 9 March 2007 (UTC) 8. "Materials, preparation, procedure, analysis" sections read like a how-to copied directly from an introductory level lab manual. Dr d12 00:29, 7 March 2007 (UTC Indeed, I'm not sure this detail belongs in wikipedia, 2 above is much more important. TransControl 09:35, 9 March 2007 (UTC)

Last edited at 09:35, 9 March 2007 (UTC). Substituted at 14:09, 1 May 2016 (UTC)