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Author: Subject: Extraction of DNA
Esplosivo
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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).

Esplosivo
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Plant DNA extraction

I found a DNA extraction from plants which does not require homogenization in liquid nitrogen. It requires centrifuging. I must buy a centrifuge soon damn it. The buffered detergent solution can be replaced by a pH buffer of approx 8.2 and some detergent. SDS binds to proteins, making them insoluble. Therefore in replacing SDS one must use a protease I guess. SDS is also the detergent.

Attachment: Edwards1991_DNA Extraction Protocol.pdf (50kB)

chemoleo
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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]

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Esplosivo
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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
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Here is another link for DNA extraction:
http://www.sas.org/MonthlyProject/SpoolingDNA/1998-09-body.h...

Sci Am. the amateur scientist column had some good articles for this area.

1) Dna extraction
2) PCR
3) electrophoresis

They used to be online but I can't find them now. If I find the articles I try to scan them.

chemoleo, you seemed to really be into this. Is this your educational background? I have a few ideas for experiments I would mind talking to you about.

Hulk
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The complete collection of Amateur Scientist articles is in a .zip file in the upload directory on Axehandle's FTP site.

PGP Key and corresponding e-mail address
chemoleo
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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.

Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
Hulk
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Thanks Polverone, I would have tried to find these Sci Am articles for the group. Which would have been a waste.

Is there a list of what is available so that someone new does not repeat such information.

Hulk
Hulk
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Hi Chemoleo

I just started to read up on PCR I saw an article in the Amateur Scientist and figured if they can do it so can I. About getting the reagents I have not tried yet.

Here is a web site that talks about building different equipment for this type of lab.

http://www.nexusresearchgroup.com/technical_data/carter.htm

Now that I am back I will post some biotech ideas I want to work on and see if anyone else has done any of this.

Hulk
Esplosivo
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The reagents used in biochem, especially in the field of molecular biology and the sort are not easy to obtain OTC, most are not available at all. The main problem of purchasing such chemicals is that they are pretty expensive. It would be interesting to hear about some of your projects.

Theory guides, experiment decides.
Taaie-Neuskoek
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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]

Never argue with idiots, they drag you down their level and beat you with experience.
denatured
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Hi All

I have a question which i hope not to be very silly ... after we have extracted DNA , what we can do with it ? can we apply the famous DNA test , that identifies every living organism?
Esplosivo
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Which famous DNA test are you referring to? DNA profiling? It depends on the protocol by which the DNA is extracted really, but as long as you have the DNA and the required electrophoresis unit, DNA probes, etc... you can do anything you want with it. The problem is DNA probes, gels, etc... are expensive and must be bought from a lab supplier.

Theory guides, experiment decides.
denatured
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Hi All

I meant the test used to identify criminals that carried out by the police
Esplosivo
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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 ?

Theory guides, experiment decides.
denatured
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Hi All

Now after what you said , we better put the extracted DNA in the oven and pour some hot chili sauce and happy meal...

By the way ... how do they measure the DNA strand length?
Esplosivo
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It is known as DNA sequencing, where apart from knowing the length one also determines the sequence of nucleotides in the strand.

[Edited on 29-4-2005 by Esplosivo]

Theory guides, experiment decides.
JohnWW
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The only problem with use of DNA sequencing and matching by the Pigs to identify alleged criminals is that DNA evidence is easier to fake than almost any other type of forensic evidence, especially where DNA evidence is collected from suspects (in some countries and for some offenses, compulsorily). All the Pigs have to do in order to frame someone they do not like for crimes they did not commit, especially rape and murder, is to plant some of a collected DNA sample of a suspect at the scene, or on the clothes or body of the victim; or plant some of the victim's DNA in the house or other property of the suspect.
sparkgap
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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 (^_^)

"What's UTFSE? I keep hearing about it, but I can't be arsed to search for the answer..."
ayush
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Few Books on DNA

12 DNA structure

 Code: http://courses.biology.utah.edu/jorgensen/2030/book%20pdf/12%20DNA%20structure.pdf 

The role of the adenovirus DNA binding protein in dna Replication and Recombination

 Code: http://mamb.ru/lib/topol/the%20role%20of%20the%20adenovirus%20dna%20binding %20protein%20in%20dna%20replication%20and%20recombination%20%20bastiaan%20van%20breukelen.pdf 

The Double Helix A Personal Account of the Discovery of the Structure of DNA

 Code: http://www.megaupload.com/?d=43V2HSV8 

The Miracle of Creation DNA

 Code: http://rapidshare.de/files/3327580/the_miracle_of_creation_dna.zip.html 

[Edited on 7-8-2005 by chemoleo]

ayush
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DNA Microarray Data Analysis
edited by Jarno Tuimala and M. Minna Laine (CSC, the Finnish IT center for Science,2003,161 pages)
ISBN 952-9821-89-1
This guidebook was a collaboration between several Finnish researchers from different universities and research institutions.The purpose of this book is to serve as course and teaching material to introduce basic concepts of microarray data analysis.Each chapter has a section on suggested reading, which introduces some of the relevant literature. Several chapters also include data analysis examples using GeneSpring software

http://www.csc.fi/oppaat/siru/siruwww.pdf

andresderis
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i'd that in biochemistry lab....obviously with quality reagents and chemicals
Chucklz
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PCR at Home

In the lab, PCR is trivially easy to do (most days). The home experimenter is seriously limited in the availability of reagents and thermocycler access, or so it appears. You can purify your own Taq from recombinant E. coli, but the procedure is rather compicated for basement lab usage. Everyone's best bet for PCR at home is to use the the Ready To Go PCR beads from GE (Amersham). Just add DI water, primers, template and you are off. You are free from master mix prep, enzyme storage in glycerol and all those other concerns.

You can cycle in a series of waterbaths. You should use the most accurate thermometers available to you to measure temperature. You CANNOT use one waterbath and change the temperature. You need 3 (95C for denaturation, one at 50-60C and one 70-75C, depending on your enzyme, and primers). You also need a good stopwatch/timer.

You will probably spend an hour or two doing the cycling. Shoot for 35-40 cycles for maximum yield.
Centimeter
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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.
chemkid
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Apparently you can see DNA under the optical microscope. I will try it out ounce i learn more about Hinduism. Why do the most interesting topics come up when i am studying for tests? Anyway, i have already looked into and performed some of these experiments. I would like to learn how to decompose the DNA and recover the ribose sugars from it.

Chemkid (craming)

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 Sciencemadness Discussion Board » Special topics » Biochemistry » Extraction of DNA Select A Forum Fundamentals   » Chemistry in General   » Organic Chemistry   » Reagents and Apparatus Acquisition   » Beginnings   » Responsible Practices   » Miscellaneous   » The Wiki Special topics   » Technochemistry   » Energetic Materials   » Biochemistry   » Radiochemistry   » Computational Models and Techniques   » Prepublication Non-chemistry   » Forum Matters   » Legal and Societal Issues