SUBJECT: Sources of DNA for isolation
Date: 11/95
It's that time of year again. Time for the DNA lab. What source of
DNA do others use. I like to have my students spool the DNA on a
glass rod because of the neat-o, gee-o effect. I find E.coli DNA does
not always spool although it is an easy source since I can grow my
own. My background has been in avian molecular systematics and I know
that bird blod is an excellent source tought I can't always get out to
collect Hose Sparrows. Most lab manuals suggest schelping to a
slaughter house to get fresh spleen etc. Is that what most people do?
Thanks for any help or suggestions.
James L. Ingold
LSU-Shreveport
Department of Biological Sciences
One University Place
Shreveport, LA 71115
(318)-797-5236 (Office)
(318)-797-5230 (Fax)
jingold@pilot.lsus.edu
Fresh calf thymus (sweetbreads) is a rich DNA source for spooling,
if you know a friendly local butcher or slaughterhouse.
We have also had above-average spooling success with pureed banana,
which is quite inexpensive. Standard protocol.
* * * * * * * * * * * * * * * * * * * * * * * * * * *
Roy Hurst _____
Assistant Professor of Science Education
The University of Texas - Permian Basin
4901 East University Blvd.
Odessa, TX 79762
Email: hurst_r@gusher.pb.utexas.edu
Phone: (915) 552-2132
Fax: (915) 552-2150
1. Two other common sources of error with E. coli DNA extraction are (a)
too few
cells and (b) the cells aren't lysed.
2. Another bacterium is Halobacterium. It's easy to lyse just using distilled
water.
3. Really CHEAP sources of DNA are at the supermarket: spinach and thymus
(the
butcher calls the latter "neck sweetbreads. Personally, this is the
only reason
I can think of that a supermarket would sell these.)
4. If you have trouble spooling, again probably because the concentration
is too
low, you might spike one of the buffers with herring sperm DNA (Sigma Chem.).
The students don't have to know.
Christine Case
Skyline College
case@smcccd.cc.ca.us
In the molecular biology lab I started this year we spooled pea seedling
DNA very nicely. The protocol for growing peas and preparing DNA we
used in in "Biotechnology Theory and Techniques" by Jack Chirikjian
(Jones and Bartlett 1995). The students seem to like this book -
obviously this is the first time we have used it.
----------------------------------------------------------------------
Walter Ogston ogston@hobbes.kzoo.edu
Department of Biology Phone: (616)337-7010
Kalamazoo College Fax: (616)337-7251
Kalamazoo, MI 49006-3295
You can't get cleaner or better DNA than with E. coli. Check the protocol
you're using. I even have my 6th graders, in an "outreach" program
grow
their own E. coli and isolate DNA, and most of them get spoolable stuff.
I'm not in the office now to get you a reference, but I published a simple
protocol that doesn't use phenol in the American Biology Teacher around
1990, titled "A Simple and Safe Genomic DNA Isolation"; by Robert
Moss.
I'm sure there are many other protocols around that work well too.
Give it another try!
-Bob Moss
Wofford College
MOSSRE@WOFFORD.EDU
I am surprised that no-one jhas mentioned onion. It seems to have become
one of the standard materials for DNA extractiuon/spooling because:
1. It is easy to obtain
2. It produces buckets of DNA
3. Works with the standard protocol.
Cheers,
Peter Gardiner
Biology Dept.
St. Michaels University School
Victoria
British Columbia
CANADA
V8P-4P5
pgardine@cln.etc.bc.ca
We use pea embryos that have been soaked overnight in water.
This process only takes 20 minutes, it can be all be done in
microfuge tubes, and it can be done with either 2M NaCl or
chloroform/octanol. It gives great globs of DNA which are
clean enough to be cut by restriction enzymes, and can quantified
by the diphenylamine method.
James M. Bader
Asst. Director, Center for Biological Education
Case Western Reserve University
jxb14@po.cwru.edu
Hi there,
We use lyophilyzed plant tissue that is provided by one of the
research labs for our DNA spooling. I have also used spinach, but I have
never tried to spoll it. I too, am looking for alternatives. So any
suggestions would be much appreciated.
I would also be interested in seeing the protocol that you use. I
have concerns about the chemicals used to extract the DNA. I have 6 labs
with 24 students each. That means alot of tissue and alot of C-tab buffer
as well as chloroform/octanol.
Thanks
Kirsten
kmahovl@uoft02.utoldeo.edu
I'm sure there are many good DNA extractions out there that don't use
phenol. Mine uses chloroform-isoamyl alcohol, and high salt.
MOSS, ROBERT "SIMPLE & SAFE GENOMIC DNA ISOLATION"; American
Biology
Teacher, v. 53, pg 428 (1991).
-Bob Moss
Wofford College
MOSSRE@WOFFORD.EDU
Hello biolabbers,
One source of DNA that spools beautifully is dog testes. These
can be collected from a local veterinarian, frozen and sliced for the
extraction procedure. The procedure uses a cold tissue buffer, 95% EtOH,
20% SDS and @M NaCl. The original protocol was written by Skinner, Laird,
Black and Clark and published by BSCS in 1970.
This content in no way reflects the opinions, standards, or policy of
the United States Air Force Academy or the United States government.
*****************************************
Helen Pigage
2354 Fairchild Drive
USAF Academy, CO 80840
(719) 472-4175
PigageHK%DFB%USAFA@dfmail4.usafa.af.mil
*****************************************
Thanks Mary and to all those gave such great suggestions for DNA.
Has anyone tried taking the spooled DNA from of any of the afore
mentioned sources, cutting it in an attempt to create an insert into a
plasmid? Right now we are attempting a similar feat in Genetics. (6 labs
of 24 students) We are using pBR322 as the plasmid and corn genomic DNA,
as
the insert.They ar both cut with BAM H1 and ECO R1 and a mixture of the
two
enzymes. The plasmids are transformed into E.coli and grown on selective
media. The plasmids which do not contain the insert will grow on a
tetracycline plate. The insert disrupts the tet gene site.
Kirsten
kmahovl@uoft02.utoledo.edu
Several people asked for this, so I'm just going to post it. The
procedure was brought to our lab by an adjunct instructor who had learned
it at a workshop. I'll try to get more information about the source. One
important tip: keep the alcohol ice cold. I store it in the freezer and
then keep it in an ice bucket during the labs.
-- Mary
------------------------------------------------------------------------
Procedure for the instructor:
Briefly blend a coarsely chopped onion with 100 ml of soap/salt
solution (100 ml Woolite, 15 g NaCl, 900 ml deionized water). Pour into
a
beaker and place in an incubator at 60o C for 15 minutes. Any hotter will
denature the DNA. Any longer and the DNA will begin to break down.
Cool mixture in an ice water bath for 5 minutes. This slows the
breakdown of DNA by enzymes. Filter the mixture through about 4 layers of
cheesecloth in a funnel placed in a beaker.
Procedure for the students:
Place 6 ml of the onion filtrate in a test tube.
Immediately and very carefully pour 9 ml of ICE cold ethyl
alcohol down the inside of the slanted test tube. Slant the tube to
reduce the mixing of the 2 liquids. The ethyl alcohol will form a clear
layer on top of the onion filtrate. Let the ethanol sit for 2-3 minutes
without disturbing it. Bubbles will form and you can watch the DNA
precipitate out of solution. It will be a white color. Gently place the
glass stirring rod in the test tube without stirring. Roll the stirring
rod between your fingers and begin to wrap the DNA around the stirring
rod. It will look like white threads around the stirring rod.
---------------------------------------------------------------------
From: Mary Farmer <mfarmer@emh1.otc.cc.mo.us>
I ran into this technique at an NABT workshop put on by
the Cold Springs Harbor folks. We use the same protocol,
substituting banana for onion - less odor and good DNA.
-- Roy
From: hurst_r@gusher.pb.utexas.edu (Roy Hurst)
I feel that there is an underlying issue which needs to be disucssed.
Let me preface my remarks by stating that I have always freely
distributed lab exercises which I have developed and have freely
utilized good ideas from others. However, I have also been burned and
"threatened" by a publisher when distributing my course lab manual
which contained some verbatim passages from a published text. [The
source was listed, but the passages were not indicated as direct
quotes]. Having had this problem brought to my attention, I have
spent some time thinking about proper citation and credit in
laboratory exercises.
It has come to my attention that the Onion DNA exercise has been
published in a book by the NABT. I don't have the exact reference.
Perhaps someone could provide it.
I feel strongly that we need to ensure that our colleagues who take
the time and effort to put together exercises that are novel (and
actually work) should get the credit for their time and effort. Also,
for many colleagues, their recognition for this type of work needs to
feed back for promotions, merit increases, and yearly reports. I am
much more careful now to include citations when asked to provide
protocols and recipes for other lab instructors. It is more than a
professional courtesy, it is a professional obligation.
Anyway, that's my opinion. I don't feel that proper atribution need
slow the exchange of good ideas and labs, but could help one of our
colleagues in a tangible way.
John
John Markwell Phone: 402-472-2924
Dept. Biochemistry FAX: 402-472-7842
University of Nebraska Internet: markwell@unl.edu
Lincoln, NE 68588-0664
Kirsten Mahovlich has a good point about what to do with the DNA
once you have prepared it. Cloning it is neat, but genomic DNA
from onions or just about anything large enough to pick up is
rather boring to clone because it is so complex, and a few
clones picked at random don't correspond to anything
interesting.
I am at the moment running a molecular biology course in which
we are trying to clone bits of chloroplast DNA, but I am not
very sanguine about getting it to work - the DNA we prepared
does not look very good when cut with the restriction enzyme.
My favorite to clone would be a large-ish virus, for instance a
herpesvirus at 100-200 Kb would be just right. When you
restrict the viral DNA you can see separate fragments, and there
is a hope of recognizing them when they turn up in a clone. The
problem here is that most of us are not in a position to prepare
decent quantities of herpesvirus DNA.
But I think of a plant virus, such as cauliflower mosiac virus
that has a DNA genome. It shouild be easy to get a ton of this
- from the supermarket if you know what to look for :) And
there is no biohazard involved. (My favorite hepesvirus infects
woodchucks and is also non-hazardous to people). Does anyone
there in labland know about getting DNA from cauliflower mosaic
virus, or another like virus?
----------------------------------------------------------------------
Walter Ogston ogston@hobbes.kzoo.edu
Department of Biology Phone: (616)337-7010
Kalamazoo College Fax: (616)337-7251
Kalamazoo, MI 49006-3295