Improving DNA extraction yield & purity?

We've got DNA extraction coming up in Semester 1, and the practical involves extraction from onion with NaCl/SDS/isopropanol, then running the DNA on an agarose gel and comparing untreated DNA, shear damaged DNA and Hinf1 digestion. I had a run through of it yesterday and the yield was really low, I only got a total yield of about 0.5ug and the A260:280 ratio was only 1.4 so I'd love to find some simple ways to improve the yield and purity.

I've spent years doing DNA and RNA extractions but they were always with commercial columns etc, I've got very little experience with this kind of manual, basic chemistry type extraction. I know that one big source of both low yield and contamination is just the technique of pipetting off the DNA layer between the isoprop and lysis buffer, so I'll practice that a bit. I also want to try soft fruit rather than onion.

What I was wondering was is there, for instance, another simple precipitation & alcohol extraction step that could be added without too much extra prep & effort?
 
We don't bother with anything other than mixing a squashed strawberry with a saline detergent blend, gentle warming, filtering into a boiling tube then adding ice cold ethanol slowly down the side of the boiling tube, a wispy material can be seen where the two liquid layers meet. I suppose more strawberries and better technique would give a better yield, if the wispy material is rich in DNA.
 
It sounds like you're trying to do electrophoresis/southern blotting almost.
To be able to see the DNA you better do the strawberry squashed experiment like Andrew Symonds said above, or you can try use strawberries instead of onion for your prac to see if it works better?
 
We've got DNA extraction coming up in Semester 1, and the practical involves extraction from onion with NaCl/SDS/isopropanol, then running the DNA on an agarose gel and comparing untreated DNA, shear damaged DNA and Hinf1 digestion. I had a run through of it yesterday and the yield was really low, I only got a total yield of about 0.5ug and the A260:280 ratio was only 1.4 so I'd love to find some simple ways to improve the yield and purity.

I've spent years doing DNA and RNA extractions but they were always with commercial columns etc, I've got very little experience with this kind of manual, basic chemistry type extraction. I know that one big source of both low yield and contamination is just the technique of pipetting off the DNA layer between the isoprop and lysis buffer, so I'll practice that a bit. I also want to try soft fruit rather than onion.

What I was wondering was is there, for instance, another simple precipitation & alcohol extraction step that could be added without too much extra prep & effort?
I was about to post a link someone put on another previous thread on this, only to discover that YOU put the link to the article! :laughing:
 
If its just the yield, and there are no students with allergies, strawberries are more polyploidy (if that is a word?)
 
I was about to post a link someone put on another previous thread on this, only to discover that YOU put the link to the article! :laughing:
Holy crap I forgot about that and my forum search-fu failed me :laughing:

Edit: OK, read the paper I'd linked and it uses liquid nitrogen, CTAB and chloroform, none of which we'd be able to use here really :(
 
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We've got DNA extraction coming up in Semester 1, and the practical involves extraction from onion with NaCl/SDS/isopropanol, then running the DNA on an agarose gel and comparing untreated DNA, shear damaged DNA and Hinf1 digestion. I had a run through of it yesterday and the yield was really low, I only got a total yield of about 0.5ug and the A260:280 ratio was only 1.4 so I'd love to find some simple ways to improve the yield and purity.

I've spent years doing DNA and RNA extractions but they were always with commercial columns etc, I've got very little experience with this kind of manual, basic chemistry type extraction. I know that one big source of both low yield and contamination is just the technique of pipetting off the DNA layer between the isoprop and lysis buffer, so I'll practice that a bit. I also want to try soft fruit rather than onion.

What I was wondering was is there, for instance, another simple precipitation & alcohol extraction step that could be added without too much extra prep & effort?
If you want high quality, high molecular weight genomic DNA, classical gDNA extractions typically involve a proteinase K digestion followed by a PCI extraction, and ethanol precipitation. This would happen in a research environment, and not in a school scenario.

A work around for students might be to get them to do the extraction and quantitation that you have done. Explain to them the limitations of the protocol (low yield, low purity etc.), and to do any further work with something like salmon/herring sperm DNA (can be sourced from Promega) that you can run on a gel in the high molecular weight, sheared, and digested forms.
 
I'm looking into giving proteinase K a go, just not sure if there's time in the session to add that to the protocol. Can't really do an ethanol extraction unfortunately since pure ethanol is a controlled substance here and it'd be too much of a hassle to use it.

For context, I've worked as a research technician most of my life, specifically in molecular biology, but I've been working as a teaching tech for the last 12 months or so (which I love). I'm in a HE institution and I suppose I wanted to try and improve the quality of our molecular practicals fairly simply. I would love if we could get the students using things like Qiagen columns etc, but the budget just isn't there :(
 
What tissue are you using?

If it something like a soft fruit, freeze thawing and mash prior to PK digestion would help. I used to digest (not plant tissue) O/N at 50/55C on a dry block in 1.5ml epps, but that would add significantly to your protocol. Phenol:chloroform cleanup is also worth avoiding - good job Qiagen, NEB etc. offer a nice selection of column based alternatives.....
 
They've been using raw onion previously, at which I raised an eyebrow. Definitely going to suggest strawberries or kiwi. Hopefully I'll be able to borrow some proteinase K and find time tomorrow to have another quick test.
 
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