Isolamento de DNA genômico de Tails rato

I realize you're using lab animals, but is it really proper protocol to punch ears as a method of identification? What about tagging with a colored bracelet? I would this that's more civil and easily noticable than making holes in ears.

CJ, I am not s scientist myself, but I found this guildeline on the UC Davis' site. They cover three techniques: tail clipping, ear punch, and toe clipping.

For toe clipping, for example, they say "Toe clipping, as a method of identification of small rodents, should be used only when no other individual identification method is feasible...  Toe clipping has the potential to induce pain and distress, alter the animal’s gait and ability to feed."

By comparison, for ear clipping, they say "Ear punch samples collected on animals do not require the use of anesthesia or analgesics, however, for identification purposes the animal must be appropriately restrained to ensure proper technique."  The document also goes on to say that instruments must be properly disinfected between mice.

So it looks to me like studies have been conducted with respect to the level of discomfort caused to animals by ear clipping and the practice is humane.  Having said that, I would be curious what studies have been conducted to measure the level of discomfort caused, as well as some explanation of why this method of tagging may be preferred over alternatives.  A cursory look on PubMed and Google didn't yield any results :\

Bracelets won't work, the mice will get rid of that very soon by chewing it off. The alternative is an ear-tag with a metal clip, which is most likely same amount of stress than ear punching.

Regarding the level of discomfort: Mice show a physiological stress response (induction of stress hormones and stress induced hyperthermia) quite easily, even if they are just lifted and put back in the cage. So it would be pretty hard to measure the stress coming from the punch, when they are already "stressed" after restraining them.

What I would not recommend though, is to tail and ear punch/cut the mice when they are that small! The mouse in the video looks like P10 (10 days after birth), if not younger. When you cut off a small piece of the ear there, it will be half the ear in the adult that is missing. And of course they can't show any real signs of discomfort at that age either (they can't really squeek as adult mice, or even bite), so it is kind of pathetic to say "See, how painfree that was". In my institute it is even forbidden to tail or mark them at this age.

thanks for this video! I'm starting today in a lab and this is one of my main responsibilities so it was nice to see a video before going :)

Thanks a lot for posting this useful video, but a question just popped in my mind: have you used the genomic DNA to amplify big fragments (2,5kb - 5kb) through RT-PCR? I've benn looking into genomic DNA extraction to use it as template for some big amps I'm starting to do...

Thanks again,

Claudio Cortés, University of Chile

BTW: in step 2 it would be 720 ul and 30 ul, right?...just a heads up

Same for step 7, 720 ul...

Im interest with this video, Can I get this video?

I was in a lab where this procedure was quite popular, but one thing was done differently. After adding isopropanol and mixing the content of the vial to precipitate DNA, it is quite easy to take the disposable tip and move floating precipitate to the vial containing 70% EtOH (this precipitate is quite sticky and quite easily sticks to the tip). Then You only need to mix it once again to wash the precipitate and then centrifugate it. This way You have one centrifugation less and the floating, non-centrifugated precipitate is much easier to wash then the pellet.

I would like to thank Dr. Zangala for this presentation.  As a bioinformatician with a background in computer science and mathematics, my job is to work on the sequence data resulting from genomic DNA extraction, though in over five years of experience in this field, I have never once extracted DNA or sequence information myself.  This was quite informative for me.

J.B. Brown, Kyoto University, Japan