Shearing DNA
Ian A. York
I've purified some DNA that's larger than 30,000 bp. I want to shear it
down to fragments of between 1000-5000 bp. (For various reasons I want to
do this mechanically, rather than using restriction ensymes.) I've been
trying passing the DNA through a needle, but I seem to be having trouble
getting the right size out. For example, passing the DNA through a
26-gauge needle 10 times produces DNA that seems smaller than the
original, but it's still >10 kb. But passing through a 28-gauge needle 4
times produces very small fragments, <500 bp.
As you can imagine, I don't have an infinite amount of this prep for
trial-and-error shearing. Does anyone have a guaranteed, tested and
consistent procedure? Or is trial and error the only way to go?
Ian
--
Ian York (iay...@panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
Karl Fischer
In article <5kj135$s...@panix.com>, iay...@panix.com (Ian A. York) wrote:
Ian,
Have you considered (though you've likely tried these) generating
fragments using either a) sonication (titrated by time at a constant power
setting) or b) DNAse I (once again time titrated against a defined amount
of enzyme)???
Just some Monday morning thoughts....
karl the hepB guy
--
Karl Fischer
ty...@bones.biochem.ualberta.ca
Jun-Zhi Wei
Hi, Ian:
I sheared some DNA samples by autoclaving. I used 121 degrees for 15
minutes to get fragments with around 500 bp. You may try shorter time to
get 1000-5000 bp.
Good luck.
J.Z. Wei
jw...@sfu.ca
ed
Ian,
why don't you try nebulizing the DNA ? someone in this department uses
nebulization very frequently and has his system finely tuned to generate a
median fragment size of about 3 Kb. He uses it for making shotgun
libraries for large scale sequencing. If you are interested, drop me a
line and I can pass this person's e-mail address to you. The procedure is
so foolproof that we used it on an undergraduate molecular biology lab :)
cheers,
Ed
Chromosome Terror
You can shear the DNA by boiling it for 10 minutes, or autoclaving it.
Doing this you'll go from a tight band to a smear in a gel. The longer you
boil it, the smaller the fragments. This is how I usually shear my DNA.
Well, not mine, but the one I work with...
Nach
eric anderson
In article <Pine.A41.3.95.970505...@aix1.uottawa.ca>, ed
<s53...@aix1.uottawa.ca> wrote:
> Ian,
>
>
> why don't you try nebulizing the DNA ? someone in this department uses
> nebulization very frequently and has his system finely tuned to generate a
> median fragment size of about 3 Kb. He uses it for making shotgun
> libraries for large scale sequencing. If you are interested, drop me a
> line and I can pass this person's e-mail address to you. The procedure is
> so foolproof that we used it on an undergraduate molecular biology lab :)
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
actually, foolproof is when it works in the hands of a rotation grad
student _AND_ a postdoc.
eric
>
--
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY 10021
(212) 639-2977
e-and...@ski.mskcc.org
NOTE: legitimate replies please remove ".nospam" at the end of the address in the header.
Bernard Murray
In article <e-anderson-06...@news.ski.mskcc.org>,
e-and...@ski.mskcc.org.nospam says...
>
>In article <Pine.A41.3.95.970505...@aix1.uottawa.ca>, ed
><s53...@aix1.uottawa.ca> wrote:
>
>> Ian,
>>
>>
>> why don't you try nebulizing the DNA ? someone in this department uses
>> nebulization very frequently and has his system finely tuned to generate a
>> median fragment size of about 3 Kb. He uses it for making shotgun
>> libraries for large scale sequencing. If you are interested, drop me a
>> line and I can pass this person's e-mail address to you. The procedure is
>> so foolproof that we used it on an undergraduate molecular biology lab :)
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>actually, foolproof is when it works in the hands of a rotation grad
>student _AND_ a postdoc.
>eric
Noooo.... The ultimate in foolproofness is when it can be done by
a visiting clinician (who doesn't try and delegate it to a nurse).
Overconfidence is a far greater problem than technical incompetence.
Oh, I have some horror stories...
Bernard
Bernard Murray, Ph.D.
ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
David L. Haviland, Ph.D.
At 09:34 5/6/97 -0500, eric anderson wrote:
>In article <Pine.A41.3.95.970505...@aix1.uottawa.ca>, ed
><s53...@aix1.uottawa.ca> wrote:
>
>> Ian,
>>
>>
>> why don't you try nebulizing the DNA ? someone in this department uses
>> nebulization very frequently and has his system finely tuned to generate a
>> median fragment size of about 3 Kb. He uses it for making shotgun
>> libraries for large scale sequencing. If you are interested, drop me a
>> line and I can pass this person's e-mail address to you. The procedure is
>> so foolproof that we used it on an undergraduate molecular biology lab :)
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>
>actually, foolproof is when it works in the hands of a rotation grad
>student _AND_ a postdoc.
Eric:
Umm... are you serious??? A nebulizer? That noisy machine that
aerosolized albuterol for various members of my family when the pollens and
molds get too high?
Would you kindly elaborate?
Many thanks in advance,
David
=============================
David L. Haviland, Ph.D.
Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX 77030
Internet:"dhav...@imm2.imm.uth.tmc.edu"
Voice: 713.500.2413 FAX: 713.500.2424
------------------------------------------------------
" Sometimes you're the windsheild, sometimes you're the bug."
=============================
David L. Haviland, Ph.D.
Jun-Zhi Wei
>
> Could you explain exactly how you did this? I'd like the same size
> fragments. (500bp). Thanks!
>
> --
> Susan
> http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html
>
> "To stay awake all night adds a day to your life."
> -- Stilgar (Frank Herbert)
>
Susan:
Here is the method I used.
1. Dilute DNA to desired concentration;
2. Transfer the sample to 1.5 ml microtubes (no more than 0.5 ml in each
tube);
3. Close the cap tightly (use cap locker if necessary);
4. Autoclave on liquid cycle for 15 min at 15 lb/sq.in.
5. Check with agarose gel.
Alternatively, you can put the tubes in boiling water for 45 min to obtain
around 500 bp fragments.
Hope this helps.
J.-Z. Wei
Ian A. York
Thanks to everyone who sent me suggestions for shearing DNA. In case
there's any interest, here's a summary of the replies, with some comments
from me. Since many of these protocols were suggested by multiple people,
I'll skip attributions altogether, because I'd likely mess them up.
My question was, "I've purified some DNA that's larger than 30,000 bp. I
want to shear it down to fragments of between 1000-5000 bp. ..."
These methods were suggested:
Passing through fine-gauge needle
Autoclaving
Boiling
Sonicating
Mini bead beating
Nebulizing (This is the one that has worked best)
DNase I digestion
A more elaborate protocol described in Nucleic Acids Research
24(20):3879-86, 1996
My comments and/or observations:
Since I plan to ligate the sheared DNA, I was hesitant to use the
autoclaving or boiling methods; both, I suspect, would lead to denaturing
of the DNA and inefficient ligation. In weak support of this, I tried
boiling my sample for various periods and then EtOH precipitating; after
boiling, much of the DNA disappeared. This may have been because the DNA
was degraded to very small fragments, or sheared over a very wide range
(so that it would be too dilute to see on a gel) or some other factor; in
any case I wasn't enthusiastic about trying to clone this DNA.
Someone mentioned that DNA sheared by sonication was difficult to clone
with. I didn't try this, so I can't comment.
Passing the DNA through a fine-gauge needle seemed like the simplest
method, but this didn't give me the size range I wanted. I suspect, but
did not do enough trials to prove, that the efficiency of this is very
concentration-dependent. Using concentrated DNA, two passes through a
30-gauge 1/2 inch needle sheared the DNA down to less than 500 bp; but
when the DNA was diluted 10-fold I couldn't get it to shear to less than
10 kbp. (Twenty passes through a 30-gauge needle gave a predominant band
at around 10 kbp +/-, with some smearing to lower MW. This was the best I
saw with the needle.)
I didn't try the DNase method; my main concern was that this would be
highly dependent on the DNA concentration, making consistent results more
difficult.
I didn't try the bead beater method, since I lacked the equipment.
SImilarly I didn't try the method in NAR; this looks pretty intimidating,
actually, and expensive, and since I only want to do this once or twice I
was reluctant to spend a lot of money on it if I could find a simpler way.
The nebulizer protocol was the one that worked first try and gave the
cleanest results. I don't know if this is an adaptation of a published
technique or if it's invented in the lab from which I got the protocol; I
also don't know if the lab wants to be deluged with requests, so I won't
post the name here. If anyone wants the names of the people from whom I
got this, drop me a note.
Here's how it works. The nebulizer chamber is slightly modified;
there's a strip of tape placed over the outflow (where the mist would
normally exit), and the tape is nicked in a small "X" with a scalpel, so
that the pressure doesn't build up. The pressure input has a tygon tube
attached so that it's delivered right to the bottom of the nebulizer
chamber (i.e. into the DNA solution itself). Dilute the DNA into 500 ul
of 25% glycerol, put it in the chamber, and nebulize. The protocol calls
for nitrogen at 14 psi; being the wild and crazy outlaw that I am, I tried
it just with our house pressurized air line, turning it up until a mist
was generated. I tried 5, 10, 15, 20, and 30 seconds of misting, tapped
down the liquid, collected it and EtOH precipitated it. The gel showed
beautiful smears of DNA of decreasing average size, from about 8 kbp
(range around 6-10 kbp) at 5 seconds, to average around 1.5 kbp (range
about 700 bp to 2 kbp) at 30 seconds. 10 to 15 seconds looked about
right; the protocol I was sent called for 12 to 20 seconds.
The lab routinely ligates the sheared DNA produced from this, so
although I haven't yet done this part I feel pretty comfortable with the
concept.
D. KIM
WRT list of methods to shear DNA:
One method listed was nebulization. I wonder if there is any hazard
associated with this method, since DNA delivered by inhalation of
liposomes in mice is sufficient to transfect the mouse's lung epithelial
cells with a marker gene. This was a possible method for teatment/cure
for cystic fibrosis. (I forget the citation, sorry).
I don't want to be an alarmist, but I would avoid breathing large amounts
of the DNA mist while doing this procedure.
Daniel Kim
dk...@nmsu.edu