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Author: Subject: Extraction of DNA
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[*] posted on 9-2-2004 at 09:01
Extraction of DNA


Here's a good article about extracting DNA from green split peas:

http://gslc.genetics.utah.edu/units/activities/extraction/

[Edited on 9-2-2004 by I am a fish]




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[*] posted on 9-2-2004 at 10:04


Excellent link!!
This is exactly the kind of information I would like to see discussed here.
Any other link like that will be very welcome.
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[*] posted on 9-2-2004 at 11:52


There will be soon. I meant to post heaps on this topic.



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cool.gif posted on 12-2-2004 at 15:25
More on DNA Purification - even your own ...


Check this for more on the purification of plant DNA (onion):

http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PDF/PlantSG.pdf

It essentially uses the same method as above, but is better explained and maybe a little bit more professional.
In additition, it details the purification of DNA from cress and peas!

I was thinking which organisms should contain large amounts of DNA - and what came to my mind first were BANANAS - I think they have over a dozen identical sets of full genomes per cell! Plenty of DNA!
Probably Banana peels (soft and old ones) are best, or the actual leaves.

One of the big disadvantages of the above methods is that the DNA gets sheared, i.e. broken into 100-500 kilobasepair pieces. Still pretty large, but for serious applications not so good.

I do have professional protocols on how to purify mammalian DNA (or even RNA) , without much shearing.
THey require more hard to get reagents though, such as phenol/chloroform, and proteases (although I think the soluble part of washing powders would do - as the biological washing powders of course contain proteases and lipases). In case you dont know, proteases break down proteins (proteins such as DNase, which breaks down DNA- a breakdown we WANT), and lipases break down lipids (fats).

Anyway, if there is interest to purify mammalian DNA I will happily post this.

In case you'd like to purify YOUR OWN DNA, I have a very good source - your own SPERM! :D - I am serious on this though, sperm naturally contains lot's of DNA and comparably little protein/fats. Of course this wouldn't be much use for the ladies.... but then there aren't many here anyhow :(

[Edited on 12-2-2004 by chemoleo]




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[*] posted on 13-2-2004 at 03:07


LOL, too bad blood doesnt have alot of DNA :(
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[*] posted on 13-2-2004 at 03:27


In case you'd like to purify YOUR OWN DNA, I have a very good source - your own SPERM! - I am serious on this though, sperm naturally contains lot's of DNA and comparably little protein/fats.


What exactly are you sugesting?


No, no. I don't want to hear it...




:D


edit 1- chemoleo, I'm editing this post to add that the link to the plant DNA paper is excelent. Great work, thanks.

[Edited on 13-2-2004 by Tacho]
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[*] posted on 13-2-2004 at 06:14


Lol - I am suggesting nothing :D -it's for you to interpret!
This a 'Mad Science' Discussion Board, so I thought it only fair to point out that there are human sources of DNA you can purify. Unfortunately, blood does indeed contain very little DNA (red blood platelets/erythrocytes dont contain nuclei, hence no DNA), so it is hardly a useful source. Else, you could of course have one kidney removed and purify DNA from that... probably quite an effort and considerably more painful!

PS Sorry if I offended anyone ethically/morally/religiously - but trust me there are much worse things you could get offended by :)




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[*] posted on 13-2-2004 at 07:16


Quote:
Originally posted by chemoleo
Lol - I am suggesting nothing :D -it's for you to interpret!
This a 'Mad Science' Discussion Board, so I thought it only fair to point out that there are human sources of DNA you can purify. Unfortunately, blood does indeed contain very little DNA (red blood platelets/erythrocytes dont contain nuclei, hence no DNA), so it is hardly a useful source. Else, you could of course have one kidney removed and purify DNA from that... probably quite an effort and considerably more painful!


For women who want to isolate their own DNA and think that surgery is going too far:

Another source of DNA is the soft tissue lining the inside of the cheek. The top layer of this can be scraped away quite easily with a teaspoon.

Alternatively, if you're willing to settle for DNA that is partially yours, you could ask a male relative.

Quote:

PS Sorry if I offended anyone ethically/morally/religiously - but trust me there are much worse things you could get offended by :)


That sounds like a challenge to me. :D


[Edited on 13-2-2004 by I am a fish]




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[*] posted on 13-2-2004 at 15:34


Alternatively, if you're willing to settle for DNA that is partially yours, you could ask a male relative.




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[*] posted on 13-2-2004 at 16:26


Well, I am a Fish, the problem is clearly that you won't get much of DNA this way. It is a well-known test to use the epithelial cells from the inner lining of your mouth - for DNA screening purposes. But for that purpose you need a single cell only, theoretically (and if you scrape them off it will be millions of cells). Of course, blood or even hair follicles will be enough. To purify a decent amount of DNA (i.e. an amount that's visible)- I am afraid you are left to purifying DNA from your kidney or... from sperm. Sorry there's no way round it :D - Unless you can find a way to replicate a whole genome with no mistakes (via the PCR reaction). If you do, you have your Nobel Prize guaranteed for sure :)



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[*] posted on 14-2-2004 at 03:06


Don't get me wrong, I am a fish, your post makes perfect sense! I just can't help thinking of the practical details....
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[*] posted on 21-3-2004 at 10:53


Strawberrie have much DNA to be extracted

If you're looking for something with a lot of DNA to extract i reccomend big juicy strawberries, they have been modified and bread to have far more than they origionally had to make them grow bigger, this is the source is used to exctract it from in college (using ice cold ethanol after pulvarising with detergent as i assume the posted link suggests, no time to read it)
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[*] posted on 21-3-2004 at 23:12


Both animal and plant cell contain RNA. There are different types of RNA : mRNA / tRNA / rRNA, all of which are vital in any living cell for different cell process (though mostly deal in some way or another with protein synthesis).
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[*] posted on 22-3-2004 at 15:15


I know that red blood cells are rather short on DNA, does anyone know if they contain RNA? If so then they might be a useful source of RNA to compare with DNA.
(I know it's not very much use as a comparison, but then again who needs DNA from cress?)
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[*] posted on 22-3-2004 at 15:37


Yes, red blood cells (RBCs) are anucleate, meaning they lack a nucleus, and the DNA/chromosomes it normally contains.
They thus have a short life span, about 2 months or so, and are regenerated from stem cells in the bone marrow.

As to RNA in RBCs - I am not sure whether they contain mRNAs. You'd only find mRNAs if protein synthesis occurred, but protein synthesis requires ribosomes, plut the endoplasmatic reticulum (ER). But RBCs dont have an ER, they have a single membrane containing the RBC, thus, I doubt there is protein synthesis going on - and hence there is no mRNA in RBCs.

As an interesting sidenote, the malaria parasite, (plasmodium falciparum) lives inside red blood cells, with its own genome. This genome is devoid of a number of housekeeping genes, such as genes needed for the citric acid cycle et cetera. In fact, the citric acid cycle machinery of Malaria hijacks the host's (the humans) proteins for its own energy production. That means that the original Malaria parasite did once contain the citric acid cycle genes, but over eons of evolution it lost those genes, because their respective protein products are produced by the RBCs...

PS why would you want a useful source of RNA to compare to DNA? You can pretty much read off RNA sequences from the DNA once you know the intron (non-coding)/ exon (coding) sequences....

Edit: Hermes aren't you lucky that the Edit time limit was extended to 48 hours ;);)


[Edited on 23-3-2004 by chemoleo]




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[*] posted on 26-3-2004 at 10:48


Well actually not all erythrocytes lack a nucleus. RBCs in birds and amphibians do have nuclei, containing DNA. Thats just some extra info.

I don't think that mammalian RBCs have any RNA in them, since it would be useless. mRNA and tRNA involved in some way or other in protein synthesis are not used, since RBCs do not synthesize proteins. I think that rRNA are also absent since they are useless because theses form ribosomes, which if I remeber correctly, are not present in RBCs.

If one wants to study the genome the RNA is useless. Chemoleo already stated why. Maybe mRNA study could yield some results on the specificity of protein synthesis being carried out inside a particular cell.
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[*] posted on 29-3-2004 at 02:36


I thought RNA are only present in bateria and viruses?!! OR am i wrong..:o



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[*] posted on 29-3-2004 at 03:17


Surely wrong. RNA is found in any living organism (or at least all of them if you exclude some viruses which contain only short DNA strands). Bacteria, being prokaryotes, have circular DNA and RNA. Eukaryotes, like us, have linear DNA and RNA. RNA is important in protein synthesis in a protein.

This site gives a VERY EXTREMELY BASIC idea on what RNA is and its function (just for those who do not know):
http://www.ncc.gmu.edu/dna/rna.htm
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[*] posted on 29-3-2004 at 09:09


Indeed.
However, there are also viruses that store their genetic information in RNA, not DNA.
This is unusual in itself, as all other living organisms store their information in DNA.
These RNA viruses, termed Retroviruses (such as HIV), convert their RNA into DNA once inside the cell, to be integrated into the genome.

Quote:

Well actually not all erythrocytes lack a nucleus. RBCs in birds and amphibians do have nuclei, containing DNA.


Now that is very interesting. Where did you hear that from? how about reptiles? Why wouldnt they have nuclei in their RBCs, even though they are between amphibians and birds, in evolutionary terms? ??




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[*] posted on 29-3-2004 at 10:05


Well as far as I know reptiles do not have nuclei in their RBCs. To prove what I said I made a quick search on google. Below is a quotation and the site from where I've got it.

Quote:

Erythrocytes in mammals are anucleate when mature, meaning that they lose their cell nucleus and thus their DNA. (Amphibian and bird erythrocytes have nuclei.)

http://en.wikipedia.org/wiki/Red_blood_cell

You can check other sources though. It's really intereseting ain't it. Reptiles falling in between the two in the evolution period should also have had nuclei in its erythrocytes but right now I cannot find any info about this.
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[*] posted on 29-3-2004 at 16:00
Professional Protocols for DNA purification


Ok, due to some requests, I have scanned several pages from the book Molecular Cloning - A Laboratory Manual, Volume 1, Third Edition. Also, see www.molecularcloning.com for more.

Here it goes:

Chapter 6, Protocol 1
is on the isolation of high molecular weight DNA from mammalian cells using phenol and proteinase. It specifies protocols for using cells growing in monolayers, suspensions, tissue samples (which is probably most interesting to us, i.e. pig liver), bloodcells (from freshly drawn samples), and it specifies the procedure for isolating DNA pieces of varying lengths. It even adds a quantification method for the DNA (fluorimetric)
Apart from Proteinase K (which is probably contained in washing powders) and phenol, no other special equipments or reagents are needed.

Chapter 6 Protocol 1-1
Chapter 6 Protocol 1-2
Chapter 6 Protocol 1-3
Chapter 6 Protocol 1-4
Chapter 6 Protocol 1-5
Chapter 6 Protocol 1-6
Chapter 6 Protocol 1-7
Chapter 6 Protocol 1-8
Chapter 6 Protocol 1-9

Chapter 6, Protocol 2
This method uses formamide. It achieves thorough dissociation of DNA-protein complexes, and one obtains very large DNA fragments (>200 kB). Disadvantages are that it takes more time, and the final DNA concentration is low (10 ug/ml).

Chapter 6 Protocol 2-1
Chapter 6 Protocol 2-2
Chapter 6 Protocol 2-3

Chapter 6, Protocol 3

Now this is the method described by the posts above, i.e. getting it from onions, peas, carrots, whatever. In terms of chemicals, it requires a strong denaturant (guanidinium hydrochloride), which I am sure most of you won't have. You can use 8 M urea instead, should be nearly as strong.
This is a very old method of isolating DNA (dating back to the 1930s), but yields on average 80 kB fragments (which is less than what you get from the methods above, but still a damn lot - i.e. the average gene is 300 bases long, which is much less than 80000 - go and figure!)
If you want to have an RNA free prep, and yet use the smalles amount of equipment/chemicals, this is DEFINITELY the ay to go!

Chapter 6 Protocol 3-1
Chapter 6 Protocol 3-2
Chapter 6 Protocol 3-3



Chapter 6, Protocol 6

This is the rapid isolation procedure of mammalian DNA. It requires RNases (to completely remove RNA), and proteinase K (to remove proteins).
You can use it for mushed up tissue, blood, etc. Note the amounts being used: 10-20 mg tissue! Very tiny amounts! in our case I guess we desire a generous upscaling! ;)

Chapter 6 Protocol 6-1
Chapter 6 Protocol 6-2
Chapter 6 Protocol 6-3


Enjoy!


PS : If you wonder what protocols 4 and 5 are about - they deal with DNA purifications from microtitre plates, mousetails and such... which is not so interesting to us, and neither to the mice! :D

[Edited on 30-3-2004 by chemoleo]




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[*] posted on 29-3-2004 at 21:24


Just for all those intereseted, one can convert the named protocols from animal extraction to plant extraction. This is done by adding a step during homogenization of the plant sample.

The plant sample is ground up (homogenized) in liquid nitrogen until a fine powder is formed.

The liquid nitrogen turns the cell wall from elastic to plastic by deep freezing. This allows the cell wall to be broken down easily, releasing its contents. I guess this step can be skipped if large plant sample are used, since some of the cell wall of some cell will eventually by crushed during homogenization. I have heard about using enzymes to digest the cell wall. Any ideas about the latter?

[Edited on 30-3-2004 by Esplosivo]
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[*] posted on 1-4-2004 at 10:21


Yes, Polygalacturonases (ExoPG / EndoPG) are used to cleave pectin of the plant cell wall. And I guess cellulase is used to break up cellulose.

Wow, thanks a lot, chemoleo, for taking the effort upon you to scan this!

[Edited on 1-4-2004 by ungebetenergast]
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[*] posted on 1-4-2004 at 10:51


Lately I've tried the crude DNA extraction from pea seeds. The volume of 'white substance' increased when an emulsifying agent was used, indication the presence of nucleic acids (ie. RNA + DNA).

What type of proteases should I use to remove the protein? Is proteinase K good?
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[*] posted on 1-4-2004 at 11:23


Lol, in the protocols I attached above it actually mentions to use proteinase K.
For those who don't have it, I think biological washing powders would be a good start - they contain cellulases, lipases, proteases (proteinases), but no DNases.
You could probably use this on all types of tissues, even plants.




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