Liver DNA

Jean Harris jean.harris at stonebow.otago.ac.nz
Sun Aug 10 06:43:56 EST 1997


Dear Helen,

We've used the following method I developed and found it worked very well
for our purposes.  The method will work on other tissues besides liver and
spleen.  I once used in on brain.

DTAB/CTAB Purification of Genomic DNA from Liver or Spleen

Solution 1:  DTAB lysis solution

	8% DTAB		8.0 g dodecyltrimethylamonium bromide
	1.5 M NaCl	8.8 g NaCl (58.44 g/mol)
	100 mM Tris 	1.21 g Tris base (121.12 g/mol) or 10 ml 1 M Tris
	50 mM EDTA 	1.86 g EDTA disodium salt (372.24 g/mol)

Dilute to 100 ml with DD H2O.  Adjust pH to 8.6 before final dilution to
volume.  Store at ambient temperature.

Solution 2:  CTAB precipitation solution.   0.5% CTAB; 0.04 M NaCl.

	2.5 g cetyltrimethylammonium bromide
	1.17 g NaCl

Dilute to 500 ml with DD H2O.   Store at ambient temperature.


Solution 3:  1.2 M NaCl	(Ion Exchange Solution)


Solution 4:  0.1 M NaCl; 10 mM TRIS base; 10 mM EDTA.  pH 8.0.  (STE)

	5.84 g NaCl
	1.21 g TRIS base
	3.72 g EDTA disodium salt

Dilute to 1 L with DD H2O.  pH to 8.0 before final dilution to volume.

Note:  To inhibit DNA breakdown this solution is 10X the normal STE
concentration in EDTA.  The original method used PBS and PBS may be used in
place of STE.  PBS seems to work just as well as STE.

Chloroform: Isoamyl Alcohol    24:1

Using a glass graduate cylinder, dilute 20 ml isoamyl alcohol to 500 ml
with chloroform.  Mix well and store in a glass bottle.  Do not store in
plastic of any kind.


Obtaining Spleen and Liver Samples:

Label storage tube with rat number and sex.  Kill rat.  Rinse blood from
the tissues with distilled, deionized H20.  Blot off excess H20 .  Cut
spleen into pieces of 0.05-0.2 g  Cut liver into 0.1-0.5 g pieces.  Freeze
on dry ice.  Wrap spleen tissues in foil to distinguish from liver.  Store
at -70o.


DNA Isolation Procedure:

Name or number all tubes and vials.  Put rat numbers on final tubes.  Weigh
the tissue and record the weight.

1.  Pulverize 0.3-0.5 g of liver or 0.1-0.3 g spleen in a glass/glass
homogenizer or an UltraTurrex.  To aid homogenization add a small amount
(1-2 ml) of STE.  It is important to homogenize the tissue well.

2.  Transfer the homogenate to a 15 ml polypropylene centrifuge tube.  Use
small washes of STE to completely transfer the homogenate.

3.  Dilute to 12-14 ml with STE.  Mix well.  Centrifuge 2400X g for 15
minutes in a swinging bucket rotor.

4.  Pour off and discard the cloudy supernatant.  Suspend the precipitate
well in 2 ml STE: H2O  1:1.  Obtain a good, homogenous suspension.

5.   Gradually add 4 ml DTAB lysis solution with mixing.  If coagulation
occurs try to break it up with a blue tip.

6.  Add 12 =B5l 10 mg/ml RNase.  Incubate at 68 degrees for 30 minutes.

7.  Add 6.5 ml CHCl3: isoamyl alcohol 24:1 and mix on a rotary mixing wheel
for at least 30 minutes.  Centrifuge 2600X g for 20 minutes.

Optional: Add 600 =B5l 3M NaOAc pH 5.2 before mixing with the  CHCl3: isoamy=
l
alcohol 24:1.  This seems to help precipitate the pellet later on.

8.  Transfer the viscous DTAB SN (~ 5.5 ml) into a new 15 ml tube.  Add 7.5
ml CTAB precipitating solution.  Gently invert the tube several times to
mix or mix on the rotary mixer a few minutes.  A filamentous precipitate of
DNA will form.

9.  Centrifuge at max speed for 2 minutes or less in the lab bench top
centrifuge.  The object is to bring down the DNA precipitate so the
supernatant can be poured off and discarded.  With one rapid, continuous
motion pour off the supernatant taking care not to disturb the pellet.  If
the DNA remains floating after centrifugation gradually pour off the
supernatant while retaining the DNA precipitate on the side of the tube.
Invert the tube onto a paper towel for 2 minutes to completely drain away
the liquid phase.

10.  Resuspend the pellet  in 4 ml 1.2 M NaCl by mixing on a rotary mixing
wheel.  The tube may be left on the rotary mixer overnight (O/N) to obtain
complete dissolution of the DNA pellet.

11.  Add 8 ml of absolute ethanol (2 volumes) and invert the tube until the
DNA precipitates.  Centrifuge at max speed in the lab bench top centrifuge
for 5 minutes.  Pour off the SN and wash the pellet with 2 ml of 70%
ethanol.  Spin down the washed pellet.  Remove the SN and drain the tube on
a paper towel for 2 minutes.  Eliminate all the remaining ethanol, but do
not completely dry the pellet.

12.  Dissolve the pellet in 1 ml DD H2O or TE.  If necessary, mix on a
rotary mixer O/N to completely solubilize the DNA.  The solution will be
viscous.  Store the purified DNA in a well-labeled 2.0 ml tube.

Notes:
1.  DTAB and CTAB may be purchased from Sigma.  CTAB is also known as
hexadecyltrimethylammonium bromide.

2.  After either CTAB or ethanol precipitation the DNA may be spooled out
and retrieved with a sealed Pasteur pipette, but it is probably best and
most efficient to spin down the DNA.

3.   Yields vary depending on how well the tissue goes into a homogenous
solution after the addition of STE:H2O 1:1 and the DTAB lysis solution and
how much of the DTAB supernatant can be retrieved.  Sometimes the plug at
the interface after centrifugation to separate the organic CHCl3 phase from
the aqueous phase is disperse and occludes some of the DNA, greatly
decreasing the yield.   When this occurs further centrifugation will not
remedy the problem.  The best way to avoid this problem is to gently but
thoroughly mix the DTAB and the chloroform: isoamyl alcohol on a rotary
mixer which will continually invert the tube and thus mix the phases
thoroughly.  When the interface plug is compact and well-formed it is
possible to go close to the plug with a blue tip to retrieve the DTAB SN.
The DNA in the DTAB SN sometimes gives the impression of being more
concentrated close to the interface plug.

4.  If using spleen, particularly a relatively large amount of spleen, an
extra STE wash of the homogenate may be useful

5.  This methodology is based on GUSTINCICH, S., MANFIOLETTI, G, DEL SAL,
G, SCHNEIDER, C:  A fast method for high-quality genomic DNA extraction
from whole human blood.  BioTechniques  1991: 11, 298-303.
International Centre for Genetic Engineering and Biotechnology, 99
Padriciano, 34012 Trieste, Italy.  Kits based on this technology are
marketed by TALENT Srl, Via del Follatoio, 12, 34148, Trieste, ITALIA.
Tel: 040.8992224.  Fax: 040.8992257.  TALENT will not divulge any
information on the kit's components.  I  recognized that the Genomix kit
components were based on the cationic detergeants used in the BioTechniques
paper, obtained some ads for the kits and worked out the methodology
described above.  Making your own DTAB and CTAB after buying the basic
ingredients from Sigma is about 1/6 the cost of buying the kit.  You do not
have any problems with glycogen using this method, you avoid using phenol
and avoid the expense of proteinase K.  The yield is approximately 1 mg of
DNA though sometimes up to 3 mg.  ELH.

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--
Eugenie L Harris, PhD		jean.harris at stonebow.otago.ac.nz
Research Fellow
Surgery Department			Phone: +64 3 474-0999  ext. 7474
University of Otago			Home:  477-1241
PO Box 913
Dunedin, New Zealand		Fax:  +64 3 474-7622

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