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 DNA separation 
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Joined: Fri Oct 08, 2010 6:08 am
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Post DNA separation
I isolated strawberry dna by using an extraction buffer made from shampoo, salt and water and precipitated the dna in isopropyl alchohol. However when I removed the precipitated dna it was all clumped together, as I had imagined. Is it possible to uncoil the supercoiled dna once it has been extracted?


Last edited by chimeraboy on Thu Nov 25, 2010 11:50 pm, edited 1 time in total.

Fri Oct 08, 2010 8:05 pm
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Joined: Thu Jun 25, 2009 12:12 am
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Post Re: Dna separation
chimeraboy wrote:
This is a little bit unrelated to biohacking. I isolated strawberry dna by using an extraction buffer made from shampoo, salt and water and precipitated the dna in isopropyl alchohol. However when I removed the precipitated dna it was all clumped together, as I had imagined. Is it possible to uncoil the supercoiled dna once it has been extracted?


can you tell me how you did this dude? please


Fri Oct 22, 2010 1:50 am
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Post Re: Dna separation
The main idea is to first lyse the cell membranes with a detergent; in my case, I used Tersaseptic clear shampoo. Then I filtered it through a cheesecloth to filter the dna. After that I filled the glass which had the dna in it with isopropyl alcohol which caused it to precipitate. Then I spooled the dna out with a straw and put in on a piece of construction paper. The problem was that I wasn't able to uncoil it from the big mass of chromosomes.


Fri Oct 22, 2010 5:03 pm
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Post Re: Dna separation
The protocol usually includes meat tenderizer. The idea is that the tenderizer contains proteases that cut up the histones that coil up the DNA.

Alcohol denatures proteins and those tend to tangle together. It may be that you have a weave of denatured proteins in the yield. If so it's possible the tenderizer can cut them into sufficiently short strands so they won't tangle.

Pure DNA tangles together too. That's why it can be seen with the naked eye, after all the strand is just a couple atoms thick.

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Sun Oct 24, 2010 11:46 pm
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Post Re: Dna separation
Yeah, in some protocols they recommend to use meat tenderizer because it contains Papain. This method didn't recommend it; I think it is because strawberries are octaploid and they have a lot of DNA available even without very much precision in lab methods. In other extractions such as from microbes and such you require ethanol and meat tenderizer.

I think it is a good idea to use the enzyme. I am not sure whether those were denatured proteins, but nevertheless it would be a good idea to try different methods.

In microbial DNA extraction, can you substitute ethanol for isopropyl alcohol and SDS with clear shampoo?

Thanks


Mon Oct 25, 2010 2:49 am
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Post Re: Dna separation
Meat tenderiser is a good idea; I never considered that. I would've thrown the whole lot into PCR, but you're probably going to get better results by removing the histones first. Alternately, you could put it in ethanol, denature with heat and try centrifuging it, although I've never tried that with nuclear DNA.

I've never had that problem with extractions, but I've never used strawberries. It might also be physical DNA damage due to rough handling.


Wed Nov 24, 2010 7:25 am
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Post Re: Dna separation
chimeraboy wrote:
In microbial DNA extraction, can you substitute ethanol for isopropyl alcohol and SDS with clear shampoo?


Shop shampoo is pretty much guaranteed to have DNA contamination; it's just not something relevant to their quality control. (Hell, some organisms live inside soap.) You could make your own sterile soap but it's probably easier to just get lab-quality SDS. If you're running an analysis that needs to be sterile then you might want to take that into account. Also remember that shampoos have various perfumes, and things to make them less harsh.

Let me know if you try it and it works, though, because that sounds like a good way to go, budget-wise.


Wed Nov 24, 2010 7:30 am
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Post Re: DNA separation
You could also try salting out the proteins. If you add enough salt in the mixture the proteins should denaturate. The denaturation should also happen in lower salt consentration for the proteins than the DNA so you could be able to separate the two from each other.


Mon Dec 06, 2010 1:14 am
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Post Re: DNA separation
That's a good idea. It sounds cheap and effective (+2!)


Mon Dec 06, 2010 11:20 pm
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