Isolation of Genomic DNA from Mouse Tails

Published 7/29/2007
14 Comments
  CITE THIS  SHARE 
Biology

Your institution must subscribe to JoVE's Biology section to access this content.

Fill out the form below to receive a free trial or learn more about access:

Welcome!

Enter your email below to get your free 10 minute trial to JoVE!





By clicking "Submit", you agree to our policies.

 

Summary

Cite this Article

Copy Citation

Zangala, T. Isolation of Genomic DNA from Mouse Tails. J. Vis. Exp. (6), e246, doi:10.3791/246 (2007).

Abstract

Protocol

  1. Cut tail pieces (3mm) and mark ears.
  2. Add 720 ml STE and 30 ml Proteinase K (10 mg/ml stock).
  3. Incubate at 55°C on heating block and vortex every hour for 3 hours at top speed for 10 seconds.
  4. Inactivate proteinase K at 70°C for 5 minutes.
  5. Quench on ice for 5 minutes.
  6. Centrifuge tail DNA for 10 minutes at full speed.
  7. Decant into new tube containing 720 ml Isopropanol. 
  8. Precipitate DNA by inverting the tube or vortexing.
  9. Spin down DNA for 5 minutes at full speed. Remove supernatant.
  10. Wash pellet with 70% ethanol.
  11. Spin down genomic DNA 5 minutes at full speed. Remove supernatant.
  12. Allow DNA to dry for 1-2 minutes.
  13. Resuspend DNA in 100-200 ml depending on size of pellet.
  14. Place tube at 55°C for 1 hour to facilitate dissolution of DNA. 

Subscription Required. Please recommend JoVE to your librarian.

Materials

Name Type Company Catalog Number Comments
STE Buffer 100 mm Tris; pH 8.5 /5 mM EDTA /0.2% SDS /200 mM NaCl / in H2O
TE Buffer 100 mM Tris; pH 5.0 /1 mM EDTA /10 mM NaCl

DOWNLOAD MATERIALS LIST

Comments

14 Comments

  1. Many thanks for this informative presentation. I used it with my HS Biology class after we did a DNA extraction.

    Sam Clifford, Round Rock, TX

    Reply
    Posted by: Anonymous
    February 7, 2008 - 12:36 PM
  2. 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.

    Reply
    Posted by: Anonymous
    May 23, 2008 - 11:49 PM
  3. 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 tŒ clipping. For tŒ clipping, for example, they say "TŒ clipping, as a method of identification of small rodents, should be used only when no other individual identification method is feasible...  TŒ 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 gŒs 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 :\

    Reply
    Posted by: Anonymous
    May 24, 2008 - 12:27 AM
  4. 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.

    Reply
    Posted by: Anonymous
    June 4, 2008 - 1:35 PM
  5. 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.

    Reply
    Posted by: Anonymous
    June 4, 2008 - 1:45 PM
  6. 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 :)

    Reply
    Posted by: Anonymous
    June 2, 2008 - 8:24 AM
  7. 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  

    Reply
    Posted by: Anonymous
    June 26, 2008 - 2:45 PM
  8. BTW: in step ² it would be 7²0 ul and 30 ul, right?...just a heads up Same for step 7, 7²0 ul...

    Reply
    Posted by: Anonymous
    June 26, 2008 - 2:51 PM
  9. Im interest with this video, Can I get this video?

    Reply
    Posted by: Anonymous
    July 7, 2008 - 11:11 AM
  10. 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.

    Reply
    Posted by: Anonymous
    August 29, 2008 - 4:46 AM
  11. 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

    Reply
    Posted by: Anonymous
    October 20, 2008 - 8:43 PM
  12. About the volumes, you can use less of the buffer and proteinase K as long as you use an equal volume of isopropanol to the volume of the buffer. I normally use 5ul per tail. Also, for general genotyping, not for cloning DNA, you can skip the 70% ethanol wash. Just let the isopropanol dry off the DNA for around 30 min instead of 1-² min.

    The ear punch is much more accurate than ear tags because mice tend to lose ear tags or get that spot infected, as well as being hard to read. TŒing is the easiest method to read, however you must be well trained to do that method. Ten days old is the gold standard for tailing age unless you use isofluorane because the nerves are poorly developed while the mouse is large enough to not have it's tail length affected too much. The pain level I have heard quoted is that it is roughly equivalent to circumcision on a human baby. John, IACUC says, "When possible, tail clipping for genotype analysis should be performed at day 17, or
    earlier, to minimize the potential for pain and distress and because DNA yields are
    higher in more immature tissue (reference the Hankenson paper). If tail clipping is
    done before a mouse is weaned at ²1 days, anesthesia is not necessary. After ²1
    days of age, anesthesia and analgesia are required. If anesthesia cannot be
    provided to a post-²1 day animal, scientific justification is required as to why
    anesthesia and/or analgesia cannot be used and why the procedure cannot be done
    before ²1 days."

    Reply
    Posted by: Anonymous
    March 4, 2010 - 2:06 PM
  13. Oops, I meant I use 5ul of Proteinase K, 300ul of buffer, and 300ul of isopropanol per tail.

    Reply
    Posted by: Anonymous
    March 4, 2010 - 2:08 PM
  14. Can't you use the tissue from the ear tagging for DNA extraction? Would save the mouse its tail ; ) otherwise nice video.

    Reply
    Posted by: Anonymous
    May 30, 2011 - 1:53 PM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Video Stats