Sciencemadness Discussion Board

Extraction of DNA

I am a fish - 9-2-2004 at 09:01

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]

Tacho - 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.

chemoleo - 9-2-2004 at 11:52

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

More on DNA Purification - even your own ...

chemoleo - 12-2-2004 at 15:25

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]

Saerynide - 13-2-2004 at 03:07

LOL, too bad blood doesnt have alot of DNA :(

Tacho - 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]

chemoleo - 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 :)

I am a fish - 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]

Tacho - 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.




-Uncle Tom?
-Yes Sally?
-Could you help me with my science project?
-Sure thing Sally! What can I do for you?
-Could you please xxxxxxxxxxxxxxxxxxx

chemoleo - 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 :)

Tacho - 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....

Chemical Peach - 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)

Esplosivo - 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).

unionised - 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?)

chemoleo - 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]

Esplosivo - 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.

darkflame89 - 29-3-2004 at 02:36

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

Esplosivo - 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

chemoleo - 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? ??

Esplosivo - 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.

Professional Protocols for DNA purification

chemoleo - 29-3-2004 at 16:00

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]

Esplosivo - 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]

ungebetenergast - 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]

Esplosivo - 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?

chemoleo - 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.

Esplosivo - 1-4-2004 at 11:46

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).

DNA extract.jpg - 34kB

Plant DNA extraction

Esplosivo - 1-4-2004 at 11:59

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)
This file has been downloaded 3035 times


Centrifugation/N2(l) homogenisation not necessary :)

chemoleo - 1-4-2004 at 12:33

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]

Esplosivo - 2-4-2004 at 11:03

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

Hulk - 19-8-2004 at 10:50

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
4) Homemade centrifuge

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

Polverone - 19-8-2004 at 12:06

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

chemoleo - 19-8-2004 at 14:23

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 :P
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.

Hulk - 8-9-2004 at 09:15

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 - 8-9-2004 at 09:22

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 - 8-9-2004 at 11:38

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.

Taaie-Neuskoek - 20-10-2004 at 03:42

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]

denatured - 27-4-2005 at 02:59

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 - 27-4-2005 at 04:23

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.

denatured - 28-4-2005 at 22:28

Hi All

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

Esplosivo - 28-4-2005 at 22:42

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 :P?

denatured - 28-4-2005 at 22:52

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 - 28-4-2005 at 23:03

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]

JohnWW - 29-4-2005 at 18:24

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 - 29-4-2005 at 19:19

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. :P

sparky (^_^)

ayush - 7-8-2005 at 03:23

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



E.b.C link

[Edited on 7-8-2005 by chemoleo]

ayush - 16-8-2005 at 00:53

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 - 7-12-2005 at 17:49

i'd that in biochemistry lab....obviously with quality reagents and chemicals

PCR at Home

Chucklz - 1-6-2006 at 12:34

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 - 29-10-2007 at 15:12

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. :D

chemkid - 29-10-2007 at 17:17

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)

DNA Extraction and Electrophoresis

Trifluoroacetic - 6-8-2008 at 19:42

Hi guys I've been on here before under a different name but it's been a while and I forgot my user ID and password.
Anyway I thought I would let you know that I have been doing some DNA extractions. I am in the process of experimenting with running the DNA through an electrophoresis unit and staining the DNA bands with various Dyes.
I made my own gel tracking dyes and buffers and am using Agar ager from a food mart.
I do have a company that has sent me a bunch of free supplies and I do have the ability to order real enzymes, ethidium bromide, buffers, and agarose; but at this time I am using the supplies I have on hand.
right now I am experimenting with staining DNA with Nile Blue and Methylene blue Chloride. I have a bunch of pictures if anyone wants me to post them. Just let me know.

DNAExtraction

Trifluoroacetic - 7-8-2008 at 17:24



IM001703.JPG - 68kB

DNAExtraction

Trifluoroacetic - 7-8-2008 at 17:26

Here is a test run of my homemade tracking Dye.

IM001705.JPG - 64kB

mac - 12-11-2008 at 15:34

Hi Trifluoracetic,

This is my first time at the sciencemadness forums - they seem great! I just wanted to let you know that some friends and I are trying to develop and publicize (via instructables and videos at diybio.org) a DIY gelbox that is pro-grade and includes a transilluminator (blue LEDs).

We posted a fun DNA extraction instructable a week ago, and when we get the gel box working we'll post some more explaining how to build one and how to extract and purify DNA to run through it. http://www.instructables.com/id/5_minute_DNA_Extraction_in_a...

Want to help? Ping me: mac at diybio.org

Trifluoroacetic - 9-12-2008 at 18:28

I've checked out the website it's very cool keep me updated. When I get time to do more experimenting I will do the same.

Trifluoroacetic - 9-12-2008 at 18:29

I'd be more then happy to help out with the project.

chief - 10-12-2008 at 10:20

DNA-extraction for everyone, very good idea. So one could track all the litle crimes, which happen and are not provable (Who stole the letter from the postbox ? Who read my private documents? Who sabotaged this or that ? etc.) oneself.
Or: Who let's his dog shit in front of my door all the time ?

Can it be done without too special or expensive chemicals ? I may order a lot of different things, too ... but neither want to get ripped for the money nor appear in the wrong context in any databases ... ?
Besides: All those private detectives everywhere: One could establish a service for them; ... it would help a lot definately ... !
It definately should not be regulated by any law, since it's probably only lightweight chemistry and a bit of electrophoresis ... so it would be legal to offer it as a service !

At least the gel-column could be made oneself, then be adjusted via a known standard-substance (2 runs: one with standard, one without; the the scaling via the standard would be possible) ...

[Edited on 10-12-2008 by chief]

[Edited on 10-12-2008 by chief]

Trifluoroacetic - 10-12-2008 at 17:51

DNA is analyzed through electrophoresis visually by comparing the bands to molecular weight markers that are added to the first column in the gel.

It really that hard to extract DNA and purify it or even do electrophoresis with it. The chemicals that are required in most cases are easily obtained and quite cheap.

The only real problem that I can see with conducting an Actual DNA analysis is with obtaining and storing the restriction enzymes and PCR enzymes. They have to be stored at a -20 degrees and are quite expensive. But with that in mind I'd still like to buy some and do some real experimenting.

Extraction of DNA from Drosophilia

mayko - 13-10-2013 at 09:45

Here is the protocol we use at work, when we are sacrificing fruit flies at the altar of reductionism.

Reagents Required

• Bender Buffer
• 5M Potassium Acetate (KoAc)
• Phenol:Chloroform, not acidic
• Chloroform
• Ice cold 70- 85% Ethanol
• Ice cold 95% Ethanol
• Rnase-free H20

Bender Buffer Recipe
0.1M NaCl
0.2M Sucrose
0.1M Tris
0.05M EDTA (MW292.24)
0.5% SDS
pH to 9.1 with NaOH


NOTE: Unless otherwise noted, keep samples on ice. Use tubes that are RNAse and DNAse free and autoclaved pipette tips. Use aliquots of DNA-specific reagents, rather than general purpose lab reagents.

NOTE: All tube and pipette tips that come into contact with TRIZOL, Phenol:Chloroform, or Chloroform must be disposed of in the Phenol: Chloroform waste bag (in hood). Work with these in the hood when possible (inhaling any amount isn’t good for you)


0. Collect flies for small prep (1 fly, instructions in brackets [ ] ) or for large prep (12 flies) in 1.7 ml, RNAse free tube. 1 fly = 40uL Bender Buffer = 20uL 5M KoAc = 40uL phenol = 40uL chloroform = 120uL EtOH

1. Add 480uL of Bender Buffer to the tube. [40uL]

2. Grind the sample down until there are no recognizable fly bits.

3. Incubate at 65C (the sandbath) for 30 min, then let cool down to RT (~5-8 min)

4. Add 4uL of 10mg/mL RNase A [1uL], invert a few times.

5. Incubate at 37C for 15min.

6. Add 480uL 5M KoAc [20uL]. Pipette up and down to mix. Incubate on ice for 10 minutes.

7. Centrifuge at 13000x for 5 minutes.

8. DNA is in the supernatant. Move supernatant to new tube and toss the old one. Add 480uL [60uL] Phenol:Chloroform.. Shake tubes well.

9. Centrifuge at max speed for 10min at RT.

10. DNA is in the top layer. Pipette the top layer into a new tube, being careful not to disturb the interface. If this layer is discolored out cloudy, repeat steps 8-10.

11. Add 480uL [40uL] Chloroform. Close lids tightly and mix by inverting ~ 15 times.

12. Spin at 15000x for 30 seconds.

13. DNA is in the top layer. Pipette it into a new tube. Add 1000uL [120uL ] of fresh, cold

95% EtOH. Incubate on ice for 10 minutes.

14. Spin at max speed for 15 minutes @ 8C.

15. DNA should have formed a pellet. Remove EtOH (everything but the pellet!) by pipetting or just pouring off. Be careful not to lose the pellet.

16. Add 500uL[100uL] 80% EtOH, spin at 8C for another 5 minutes, then remove EtOH.

17. Spin down tube quickly and remove any excess EtOH with a 200uL pipette.

18. Let pellet air dry for 10 min at RT.

19. optional: If still smells of Ethanol, put on sand bath for 5min at 65C.

20. Elute in 52uL [20uL] EB buffer. Pipette around tube sides to elute not-visible DNA. This step can be wildly variable and depends on downstream intentions. ddH2O might be favorable.



mayko - 16-10-2013 at 10:31

A slight variation: RNA extraction from Drosophila. RNA is a little bit more fragile than its deoxyribo cousin, so a different protocol is required.

Self-assemble your own molecular computers at home!
http://www.technologyreview.com/news/410986/computing-with-r...



Attachment: RNA Isolation from Flies.doc (50kB)
This file has been downloaded 1013 times


confused - 16-10-2013 at 10:54

the thing about doing actual gel electrophoresis is that DNA stains are expensive, if you're thinking of using Ethidium Bromide, cleanup and disposal is a mess. But if im not wrong, you can also use Methalene blue for stains and gentian violet as a loading dye.

The really pricey reagents are PCR enzymes,primers, DNA standard and restriction enzymes

Little_Ghost_again - 21-10-2014 at 12:13

A lot of the above seems way out of date, when I get a chance I will write a detailed post on how to use and make a home PCR and how to sequence DNA at home, the resolution isnt great but the cost is under £30. If you have a flat bed scanner and a copy of matlab then I can also detail how to get better resolution, chopping and inserting DNA is not hard.
I have seeen this done time and again here in my dads lab, ok the equipment is different but most can be approximated enough.
If you want a complete chromosome to play around with then go for the fruit fly, really easy to separate and very large for a chromosome.

amyi - 20-5-2015 at 00:18

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.

So which kind of links do you want?

Laurylamido propyl Betain - 16-7-2015 at 00:47

DNA extraction?The moment I saw the word ,I thought of so many fiction films .

gel electrophoresis resolution

calvin - 3-12-2015 at 23:35

Has anyone here actually performed gel electrophoresis using either EtBr or other dyes? If so, what sort of resolution is possible with such techniques? Most of the images I can find online of band patterns are so poorly defined that it doesn't seem possible to actually sequence more than maybe a dozen bases this way.

The reason I ask is because our school's computer science club wants to build an automated DNA sequencer based on sanger sequencing but using digital imaging and OpenCV to read the DNA sequence. Would it be possible to sequence 50bp+ sequences this way?

NitratedKittens - 28-12-2016 at 08:13

Quote: Originally posted by I am a fish  
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]


The best way for a woman to isolate her own DNA in large quantities is to centrifuge her blood then extract from the WBC layer.
Or could the epithelial cells shedded during a period contain enough DNA.


[Edited on 28-12-2016 by NitratedKittens]

sperm DNA extract

nighthawk - 5-4-2017 at 09:13

How would you extract this DNA? Would a light microscope work to see anything?

TriiodideFrog - 2-10-2020 at 23:43

I suggest using your tongue to dislodge some cheek cells, then spitting your saliva into a test tube.

TriiodideFrog - 2-10-2020 at 23:49

You can also make homemade DNA extraction solution. Fill a beaker with 1/2 cup of water. Slowly add in 2 teaspoons of dish soap and 1/2 teaspoon of table salt. Gently mix this solution without making bubbles until the salt dissolves. Fill he test tube with saliva 3/4 with the solution. Then, ice-cold alcohol is added to the solution causes the DNA to precipitate out. The DNA will look like stands of fine silk. You can also try this experiment with crushed strawberries.