Extraction of DNA

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! - 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]

LOL, too bad blood doesnt have alot of DNA

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...







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]

Lol - I am suggesting nothing -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

Quote:

Originally posted by chemoleo Lol - I am suggesting nothing -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!

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.


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

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 - 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

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]

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

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!

[Edited on 30-3-2004 by chemoleo]

Right, I missed that. Biological washing powders also contain amylases to remove starch residues. I attached a pic showing my experiment with the control. The extraction of the DNA is quite visible. I'll try removing the proteins and carry out a DNA test (rather a nucleic acid test).

Plant DNA extraction

Centrifugation/N2(l) homogenisation not necessary :)

I don't think homogenisation in N2(l) is ultimately required, not even centrifugation. In the link I posted above (4th post from top), it gives a protocol to the purificataion of plant DNA from onions, peas, et cetera. It doesn't even require centrifugation, just filtration. Filtration can often be used instead of centrifugation, especially if the particles are >50 micrometers or so (and dont tend to form aggregates). That is, particles smaller than that are hard to filter, and require vacuum suction and such, and even then it doesn't go fast ( I know that from the various filters that I use, which go down to 20 uM).
Of course, for the professional purification of DNA, a centrifuge and proper equipment/reagents is preferred.

On the note of SDS (sodium dodecylsulphate) - it is a strong detergent that solubilises proteins, it brings even ordinarily insoluble proteins into solution! This is why it's used for SDS gel electrophoresis, where proteins are linearised (=unfolded) and solubilised by SDS, and hence their size is judged by their speed of migration in the gel (which is a function of the number of amino acids in the protein). Other solubilisers/denaturants of proteins are 8M urea or 6M guanidinium hydrochloride, which won't affect DNA either.

DNA purification kits often contain denaturants of the above kind, proteases, SDS and RNases. Plus a buffer that adjust the solution to a pH such that mainly DNA binds to an ion exchange membrane. This way I have purified hundreds of samples

PS Pls merge threads to avoid cluttering. thanks.

[Edited on 1-4-2004 by chemoleo]

Good to know urea dissolves proteins. It will proove useful when extracting DNA from the precipitated mixture. Urea is cheap , much cheaper than SDS right lol

Hulk, reactions such as PCR are pretty much impossible to do at home, and pretty much pointless - unless you want to clone toxic genes for your bioweapons program or something
But seriously, the reagents aren't exactly available OTC, even though PCR is done in labs day in and out. In addition, a PCR cycler isn't a device easy to make (rapid cooling/heating).

Anyway - if you have experiments to discuss - this is the place . There are plenty of people who may be interested, plus it's always better to get more than just one opinion/comment.

For schools there is a GFP-kit from Biorad available, its requires nothing more than sterile water...you can do everything with BASIC lab equipment. A water bath is needed althought.

The kit contains everything (including enzymes, plasmids, bacteria) I guess you only have to make the bacteria compentent - not that much work -

You need a waterbath and something to sterilise your media in. (a high-pressure pan will do) Also a UV lamp is needed to check for positive transformands.

I think a miniprep is easy a home-scale...
The only difficult thing will be the centrifuge steps, but a centrifuge is needed for almost ALL these kind of expreriments...

A PCR is easy if you have 3 water bath's...and a lot of patience. (transfering the tubes 30 cycles from one bath to another...)

Edit: I have protocols for DNA/RNA extractions out of tomato fruits, cucumber leaves and potato leaves/tubers. Also some for the isolation of Phytophthora infestans, but I can hardly imagine someone is interested

Basically it are all phenol/chloroform extractions, but some plants are harder to extract then others by a high sugercontent, or other nasty things.

For the sake of completeness: wear gloves during DNA isolation to prevent DNase activity...

[Edited on 21-10-2004 by Taaie-Neuskoek]

Then what you meant is DNA profiling. I think it is relatively out of limit to the home scientist, although with some home-made equipment (electrophoresis units can be made out of perspex, I have built a small one for demo purposes and a centrifuge can be easily made I suppose) and some probes which can be bought (they are expensive, very very) and restriction enzymes I think one could produce something similar to a DNA profile. Btw, usually satellite DNA is used in such tests. Many types can be targeted according to the type of probes one uses.

Using the DNA mixture as is and using a gel with a large pore size (say a low conc. of agarose) could be used to identify different species I think - different species have a different number of DNA strands of varying lengths, but the long chain of the DNA will make this a sloww process. The DNA strands could then be stained (many commercial stains exist).

Any more ideas anyone? What can be done with the extracted DNA? Are there any OTC sources of resitriction enzymes ?

You might fancy growing a nice batch of E. coli just for their Eco R1 endonuclease. Don't ask me about backyard-style purification however.

sparky (^_^)

PCR at Home

This is a wonderful topic!

I wanted to comment more on SDS as it has a very important aspect that was not covered. Perhaps its most important characteristic is that it is negatively charged and bonds to a protein in direct proportion to its molecular mass. Thus a SDS bound protein when run on a gel, will not experience any variability due to charge. Furthermore, as chemoleo pointed out, it disrupts a protein's tertiary structure, eliminating all variables other than size.

Another option would be to use iodoacetamide (IOA) with a dithiothreitol (DTT) quench. Perhaps these might be more easily obtained, although I doubt it.

Most labs that I've done where DNA is to be extracted just heat the cells up to ~100*C to break down the cell wall. Of course this is where the microfuge comes in handy. However you should be able to achieve the same results by letting the suspension settle over night.

As far as running a DNA comparison between two subjects, I imagine it could be done to a certain extent at home. The issue would definately be PCR. You would have to have access to many primers and even in a lab environment these tests are often quite difficult to obtain good resolution. I can guaranty you that a home brew PCR reaction is going to result in far too variable of data to make any interpretations.

I was wondering if anybody had some ideas on how to obtain one long distinct strand of DNA. By this I mean that I want a single continuous strand of DNA with no tertiary structure and no fixed modifications such as SDS. I think it would be awesome to be able to wind it up and place a spiral of your DNA in a sample vial. On that note, I am seriously having trouble thinking of any other viable methods of collecting one's cells for DNA other than via sperm.

DNA Extraction and Electrophoresis

DNAExtraction

DNAExtraction

Here is a test run of my homemade tracking Dye.

Extraction of DNA from Drosophilia

Quote: Originally posted by Tacho

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.

gel electrophoresis resolution

Quote: Originally posted by I am a fish

Quote: Originally posted by chemoleo Lol - I am suggesting nothing -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. [Edited on 13-2-2004 by I am a fish]

sperm DNA extract