|WikiProject Molecular and Cell Biology||(Rated Start-class, Mid-importance)|
Transfer of DNA fragments onto nitrocelluose paper
You've skirted over the issue a bit. To my understanding using the (older) capillary method filter paper is used to wick buffer so that it flows through the gel and carries the DNA fragments to the membrane. Effectively blotting the DNA bands from the gel to the nitrocellulose paper. Apparently nowadays Electrolytic transfer is often used as well/ instead.
^^^This. Electroblotting is faster than putting weight on on top of the membrane and allowing capillary action to take over, especially since polyacrylamide gels don't have charge, unlike groups in agarose. Typically polyacrylamide gels are favored over agarose gels because they have more consistently sized pores and the results from polyacrylamide are more reproducible. Polyacrylamide also has a higher resolving power than agarose gels (http://bitesizebio.com/20395/agarose-versus-polyacrylamide-not-all-gels-are-created-equal/). That being said, if you are screening for large rearrangements, an agarose gel would be better. And of course, agarose is safer to work with.
I feel like this article should note that UV cross-linking to the membrane takes about 10 minutes as opposed to baking for two hours. — Preceding unsigned comment added by 22.214.171.124 (talk) 14:43, 23 July 2016 (UTC)
I cannot recall a Hawaiian blot and it has no link, what is this method for detecting biochemicals? SableSynthesis 15:08, 28 March 2006 (UTC)
request for information
We should add some info so kids doing their homework can get the answers
Hey also, just thought that someone should change this portion in the methods
"The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non-specific binding of the probe."
Within the paragraph it says "most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA"
I am pretty sure that's not right, although funny.
- Hehe, as odd as it sounds, salmon sperm is indeed a common source of non-specific DNA. Sperm are tiny cells, almost as small as they can possibly be while still containing an entire genome, so that makes them excellent for extracting DNA with little contaminating material. You can search "salmon sperm" in Wikipedia to verify that it's commonly used. Adrian J. Hunter(talk•contribs) 07:33, 15 March 2011 (UTC)
Capitalization of other blots
The CDC page on writing style says that capitalized Western blot is the preferred. I'm sure one can make arguments for or against capitalizing, but usually style should conform to one standard and the CDC is a good enough authority. Calgirl2 (talk) 18:23, 29 November 2007 (UTC)
- I think that you will need to discuss this on Western blot, because this entry uses likewise "western blot" rather than the capitalized form, and so style within Wikipedia would be inconsistent. But I'd avoid flogging dead horses, as camps pro and con capitalization are firmly entrenched, and both have good arguments for making their cases. Malljaja (talk) 19:14, 29 November 2007 (UTC)
- A brief addition to my comment. I note that the CDC lists Southern and Western blot as requiring capitals, but not their methodological cousin, northern blot--this is inconsistent and may may rest on CDC's erroneous assumption (which I've heard before) that the western blot was invented by someone called "Western". Malljaja (talk) 19:20, 29 November 2007 (UTC)
Southern blot is named for E.M. Southern, who invented it, and is therefore capitalized. Other blot methods named by analogy -- e.g., Northern, Western -- are also capitalized by analogy. See Dorland's Medical Dictionary (any edition). Linguina (talk) 11:56, 2 August 2009 (UTC)
The comment(s) below were originally left at 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., and are posted here for posterity. Following
|The description of the hybridization probe seems a little incorrect to me. The probe is often made by "Klenow labeling" and random hexamer/octamer primers are used. The template is usually a plasmid fragment/PCR product thus usually two DNA strands are available as templates in the reaction. So the actual probe is a mixture of fragments - not a single specific fragment.
Moreover, the probe binds to a complementary sequence, and not one identical to it. I had a feeling this was not too clear from the article.
Last edited at 21:28, 16 May 2007 (UTC). Substituted at 06:37, 30 April 2016 (UTC)
^ I agree. The major benefit of Southern blotting is that you don't have to know exactly what mutation or rearrangement you are looking for. — Preceding unsigned comment added by 126.96.36.199 (talk) 14:44, 23 July 2016 (UTC)