DNA Extract

Genomic DNA Isolation Reagent

Cat. No.: 2065-001-DB

Store at: Room Temperature

Product Description
DNA Extract is a non-organic and ready to use reagent for the isolation of genomic DNA from samples of human,
animal, plant, yeast, bacterial and viral origin. DNA Extract is an improved version of the Chomczynski method
(1), which is based on disruption of cells in a guanidine-detergent lysing solution that hydrolyzes RNA and allows
the selective precipitation of DNA from a cell lysate with ethanol. Following an ethanol wash, DNA is
solubilized in water or 8 mM NaOH. There is no phenol in DNA Extract. The protocol is fast and permits
genomic DNA isolation from a large number of samples of small or large volumes. The procedure can be
completed in 10-30 minutes with DNA recovery of 70-100%. The isolated DNA can be used, without additional
purification, for southern analysis, dot blot hybridization, molecular cloning, RFLP, PCR and other molecular
biology and biotechnology applications.

Kit Reagent
50ml solution containing Guanidinium Isothiocyanate.

Reagent Required But Not Supplied

* Absolute Ethanol * Digestion Buffer (for mouse tail): 50mM Tris pH 8.0
* 95% Ethanol 100mM EDTA
* 8 mM NaOH (fresh preparation) 0.5% SDS
* Chloroform (for plants) * RBC Lysis Solution

Storage
DNA Extract should be stored at room temperature. However, storing at lower temperatures will cause the
Guanidine Isothiocyanate to come out of the solution. If the reagent is warmed, the Guanidine Isothiocyanate
should resolubilize instantly.

Handling Precautions
DNA Extract contains irritants. Handle with care, avoid contact with skin, and use eye protection. In case of
contact, wash skin with a large amount of water. Seek medical attention.
Protocol for Genomic DNA Isolation
1. Homogenization
All samples should be gently, but thoroughly, homogenized with the DNA Extract reagent. Homogenization
can be achieved by repetitive pipetting with a pasteur pipette. The sample will become viscous due to the
release of high molecular weight genomic DNA. Do not pipette the sample too vigorously, as this will shear
the genomic DNA. All samples should be held at room temperature for 5 minutes, unless stated otherwise.

1.1 Tissue
Gently homogenize tissue samples in the reagent. Use 1ml DNA EXTRACT per 50mg tissue.

1.2 Cells
Cells grown in monolayer - should be lysed directly in the culture dish by addition of 1ml DNA Extract per
10cm2 area of culture dish. Discard the media, add DNA Extract and pass the cell lysate several times
through a pipette.
Cells grown in suspension - use 1ml DNA Extract per 107 cells. The cells should be pelleted and then lysed.
Alternatively, use the suspension (volume < 0.1ml).
Cell nuclei - use 1ml DNA Extract per 107 cell nuclei. The nuclei can be either in pellet or suspension
(volume < 0.1ml). Mix the samples by inverting the tubes or repeated pipetting.

1.3 Bacterial Cells

Gram positive - use 1ml DNA Extract per 107 cells. Freeze cells in liquid nitrogen and grind to a fine
powder using a mortar and pestle, homogenize briefly, and gently mix for 1 hour at 60oC.
Gram negative - sediment the cells and use 1ml DNA Extract per 107 cells. Lyse the cells by repetitive
pipetting and gently mix for 15-60 minutes at 60oC.

1.4 Yeast Cells

Sediment the cells and use 1ml DNA Extract per 107 cells. Homogenize briefly and gently mix for 15-60
minutes at 60oC.
1.5 Plant
Use 1ml DNA extract per 50-200mg of plant. Freeze cells in liquid nitrogen and grind to a fine powder
using a mortar and pestle or homogenizer. Gently mix for 1 hr at 60 oC, and proceed with section 2 of
the protocol.
1.6 Liquid Matrices
To isolate DNA from liquid matrices including stool, sputum, urine, wound exudate and viral cultures,
gently homogenize 1ml sample in 10-15ml DNA Extract.
1.7 Whole Blood
To isolate DNA from whole blood, add 1ml of whole blood to 2ml RBC Lysis Solution.
Gently mix at room temperature for 5 to 10 minutes. Centrifuge at 300g for 10 minutes and
discard the supernatant. Add 1ml DNA Extract, and mix the sample by repeated pipetting.
Hold for 5 minutes at room temperature.

1.8 Mouse Tail

Add pieces (1-3mm) of mouse tail (up to 20mg) to 0.5ml digestion buffer supplemented with
400g/ml proteinase K. Incubate at 55C for 1-4 hours with mixing, or overnight at room
temperature. Briefly centrifuge the samples and transfer the supernatant to a new tube. Add
0.5ml DNA Extract to the supernatant and incubate at room temperature for 5 minutes. Add
 1ml of absolute ethanol, mix, and allow to sit for 1-3 minutes. Spool the DNA and proceed
with Step 5.

1.9 Biohazardous Material

When working with biohazardous material, Proteinase K digest can be used (see 1.8). This
technique eliminates aerosols and improves biosafety.

2. Phase Separation (PLANTS ONLY)

Add 1ml chloroform per 1ml DNA Extract. Allow to stand for 5 minutes at room temperature, and centrifuge
at 12,000g for 10 minutes at room temperature. Following centrifugation, transfer the upper (aqueous) phase
to a clean tube and precipitate the DNA by adding ethanol: 1 volume of aqueous phase with 1 volume of
ethanol. Mix the samples by inverting the tubes 10 times and store them at room temperature for 5 minutes.
Sediment precipitated DNA at 5,000g for 4 minutes and discard the resulting supernatant.

3. Centrifugation (optional)
This step removes insoluble tissue fragments, and is recommended for tissues containing a large amount of
extracellular material (liver, muscle). Centrifuge at 10,000g for 10 minutes at room temperature.

4. DNA Precipitation
Add 1ml of absolute ethanol per 1ml of DNA Extract. Mix samples by inverting the tubes 10 times. Make
sure that the DNA Extract and the ethanol make a homogenous solution. Store the samples for 3 minutes at
room temperature. DNA should become visible. Remove the DNA by spooling with a pipette tip or centrifuge
at 5,000g for 5 minutes. For small quantities of DNA use centrifugation.

5. DNA Wash
Wash the DNA pellet twice with 1ml 75% ethanol. To remove contaminants from difficult sources (such as
liver, kidney, yeast, gram positive bacteria), for the first wash use solution containing 50% DNA Extract and
50% ethanol. Suspend the DNA by inverting the tubes 10 times. Allow the DNA to settle to the bottom or
centrifuge at 1,000g for 1 minute. Remove ethanol by pipetting.

6. DNA Solubilization
Remove remaining ethanol wash using a pipette, but do not let the DNA pellet dry completely. Next, dissolve
the DNA in 8 mM NaOH (fresh preparation). Add a sufficient amount to reach your desired concentration.
Note that a higher concentration than 0.3g/l will cause a very viscous solution that will be hard to work
with. Store the sample for 5 minutes and then dissolve the DNA by pipetting. For high concentrations,
heating at 55oC will be required. For preparation from tissues or plants containing insoluble material, remove
the insoluble material by centrifugation at 12,000g for 10 minutes. The final preparation of genomic DNA
isolated with DNA Extract contains 20-100 kb with the A260/A280 at a ratio of 1.6-1.9.

Note
The resulting DNA may contain some degraded RNA. The concentration of the RNA is less than 3% of the
DNA. For most methods this is no problem. If you require RNA-free DNA, apply RNase to the DNA sample.
 pH Adjustment of DNA Samples Dissolved in 8mM NaOH
For 1ml of 8mM NaOH, use the following amounts of 1M Hepes, free acid:

Final pH 1M Hepes (µl)

7.0 42
7.2 30
7.5 18
7.8 13.5
8.0 11.5
8.4 9.5

Halting Points during Isolation

* The lysate which contains DNA Extract can be stored for:
18 hours at room temperature
9 months at 4C
9 months at -20C

* During washes, DNA can be stored in 95% ethanol for at least 1 week at room temperature, or 3 months at
4C.
* For long-term storage of high molecular DNA, re-precipitate the DNA and store in ethanol at 4C.

Reference
(1) Chomczynski, P. and Sacchi, N., Anal Biochem., 162:156-159 (1987)