3 replies to this topic

[Niverdia]

Greetings, all.

I work as a senior lab assistant at a university and one of my longer term tasks is to develop a DNA extraction protocol for medical student molecular biology class that could be completed, for the most part, within a 1,5 hour-long lesson.
I know there are plenty of easy DNA extraction protocols out there for demonstration, but the professor in charge who doesn't do any practical work themselves expect me to develop a protocol that not only works as a demonstration of a puffy cloud of extracted DNA, but which also produces DNA of sufficient quality for PCR amplification, while not using any particularly dangerous materials NOR any expensive ready-to-use DNA extraction kits. Basically, the expectation is to develop a fast, safe, effective DNA extraction protocol with cheap over-the-counter materials used for extraction and purification. I understand the enthusiasm and the educational value of being able to extract and amplify your own DNA, but this doesn't sound very feasible.

So far, I've been trying to tinker with the protocol I've added in the attachment that uses detergent and alcohols, but with a mouth rinse method I've only obtained very small concentrations of badly purified DNA. Besides the detergent, I've experimented with adding proteinase K, but considering the short time limits for the overall process, the short incubation period didn't help much either.

Can anyone suggest alternative protocols that might work, or am I right in thinking that with the given circumstances the expectations for developing such a DNA extaction protocol are very unrealistic?

Attached Files

•  McGrath_Final_draft_ETP_2009__DNA_Fingerprint_Typing_of_Bubble_Gum_.doc   451K   78 downloads

[hobglobin]

What about a boiling prep? It's fast and dirty and don't need expensive chemicals. As it is for plasmids you have to modify the protocol surely (e.g. a grinding step).
Another possibility is a salting out protocol, that works for most organisms quite well. You need a lysis buffer with Tris, EDTA, SDS and proteinase k (not sure if other surfactant is possible, and the enzyme can perhaps be replaced by meat tenderiser (papain), as some protocols with kitchen chemicals suggest), lots of sodium chloride, and alcohol for precipitation....
I think a major problem may be that many of the extraction protocols need time for lysis (can be shortened by more heat) and for DNA precipitation with EtOH on ice. The longer the better usually. No idea how to shorten this.

[leelee]

Would it have to be a human DNA extraction?

Also, you said mouth rinse- have you tried scraping buccal cells instead? We do this with our undergrads using a plastic disposable spoon, and we do get a decent amount of DNA. Enough for PCR etc, and they almost always work- although inevitably a few students will stuff up. From memory we do it in a 2 hr class, that includes setting up the PCR (but these are science students, not medical students, so they do tend to be a bit better/faster in the lab, in my experience).

But I wouldn't describe what we get as a cloud of DNA.

I'm pretty sure there are high school level protocols out there for use with things like tomatoes, or liver etc.

(also, for what it is worth, I actually think it is better for the students if they can't see or can only barely see the DNA pellets- I think it teaches them the reality of extracting DNA from clinical samples much better than the unrealistic bucket loads of DNA type demonstrations).

[pito]

From what do you want to extract the DNA?

If you need an easy/fast method to just show how it works and do a small PCR then you can use a simple Kiwi DNA extraction... This is pretty much the fastest out there to show students how it works...
In Belgium its "the method" to use with students that are new and/or no experts.

And the reason why we also use a kiwi is that its not allowed to extract your own DNA...
(to be honest, I would find it weird that you are allowed to use human/students DNA).