*Isolates high quality genomic DNA in one hour from tissues
and cells with a single monophasic reagent
*Can be used to back extract DNA from "Single Step Method " RNA Preps (i.e.,
RNAzol, RNA Bee, RNA Stat-60)
*Does not contain phenol
*RNA and proteins sequestered in organic and interphase; DNA remains in upper
DNA STAT-60, a single monophasic
reagent containing a chaotropic cell disrupter and a non-corrosive
phenol free extraction reagent, replaces cumbersome, labor intensive
methods of genomic DNA isolation. Following tissue or cell
homogenization in the DNA STAT-60, and after the addition of
chloroform, the homogenate separates into two phases: the aqueous
phase and organic phase. The DNA remains in the aqueous phase while
RNA and other cellular components, including proteins, are
preferentially partitioned in the organic phase and interface. The
DNA STAT-60 method does not require ultra centrifugation, and can be
completed in under 1 hour. The DNA STAT-60 isolates high molecular
weight genomic DNA from samples of human, animal, plant, yeast, and
bacterial origin and is particularly well suited for the
simultaneous processing of multiple samples. RNA and proteins are
sequestered in organic and interface: DNA remains in upper aqueous
phase. Protocol can be completed in 60 minutes. Single monophasic
reagent with extended shelf life. Does not contain phenol or
hazardous organics. Isolates genomic DNA from samples of human.
animal, plant, yeast and bacterial origin. Can be used to back
extract DNA from "SINGLE STEP METHOD" RNA preps (i.e.
RNAzol B. RNA STAT-60).
2. REAGENTS SUPPLIED DNA
50 ml, 100
ml, or 200 ml bottle containing clear solution of DNA STAT-60
Ready to use.
STORAGE Refrigerate at 2-8oC.
Protect from exposure to light.
STABILITY 6 months. Refer
to expiration date stamped on label.
3. REAGENTS REQUIRED, BUT NOT SUPPLIED
grade) Isopropanol (ACS grade) Ethanol (ACS grade)
DNA isolation by the DNA
method includes the
1. Homogenization DNA STAT-60 (1 ml per
50-100 mg tissue, or 5-10 x 106 cells).
Extraction 1 vol. of homogenate + 0.2 vol. of chloroform.
DNA Precipitation 0.5 vol. of isopropanol.
Wash 75% ethanol. Unless stated otherwise the procedure is
carried out at room temperature.
A. TISSUES: Homogenate tissues in
the DNA STAT-60 (1 ml/50-100 mg tissue) in a glass-Teflon or
B. CELLS: Cells grown in mono layer
are lysed directly in a culture dish by adding DNA STAT-60 (1 ml/3.5
cm petri dish) and passing cell lysate 5-10 times through a pipette.
Cells grown in suspension are sedimented then lysed in a DNA STAT-60
(1 ml per 5-10 x 106 cells by repetitive pipetting.
4.2 DNA EXTRACTION Following homogenization, add 0.2 ml of
chloroform per 1 ml of DNA STAT-60, cover the sample tightly, shake
vigorously for 15 seconds and let it stay at room temperature for
2-3 minutes. Centrifuge the homogenate at 12,000 g (max.) for 15
minutes at 4oC. Following centrifugation the homogenate
separates into two phases: a lower organic phase and the upper
aqueous phase. DNA remains in the aqueous phase whereas RNA and
proteins are in the interface and the organic phase.
4.3 DNA PRECIPITATION
Remove supernatant and
wash the DNA pellet once with 75% ethanol by vortexing and
subsequent centrifugation at 7,500 g (max.) for 5 minutes at
4oC. Add at least 1 ml of 75% ethanol per 1 ml of the DNA
STAT-60 used for the initial homogenization. At the end of the
procedure, dry the DNA pellet briefly by air drying (5-10 min.).
Dissolve the DNA pellet in water or in 1 mM EDTA, pH 7. Pass the
pellet a few times through a pipette tip. An incubation for 10-15
minutes at 55-60oC may be required to dissolve DNA
5. RECOVERING DNA FROM "SINGLE STEP METHOD" (RNAzol B, RNA
5.1 DNA REVERSE EXTRACTION
Remove aqueous layer containing
RNA. Add 800 ul of DNA STAT-60 reagent per 1 ml of RNAzol B or RNA
STAT-60 used for the initial homogenization. Add 0.2 ml of
chloroform per 1 ml of DNA STAT-60, shake vigorously for 15 seconds
and let it stay at room temperature for 2-3 minutes. Centrifuge the
homogenate at 1000 g (min.)-12,000 g (max.) for 15 minutes at
4oC. Following centrifugation the homogenate separates
into two phases: a lower organic phase and the upper aqueous phase.
DNA remains in the aqueous phase whereas RNA and proteins are in the
interface and organic phase.
5.2 DNA PRECIPITATION
Follow procedure as outlined in
6. SPECIAL HANDLING PRECAUTIONS
The DNA STAT-60
contains an irritant (Guanidinium Salts). Can be fatal. When working
with DNA STAT-60 use gloves and eye protection (shield, safety
goggles). Do not get on skin or clothing. Avoid breathing vapor.
Read warning note on bottle.
1. Chomczynski, P. and Sacchi, 1987. Single
Step Method and RNA Isolation by Acid Guanidinium
Thiocyanate-Phenol-Chloroform Extraction. Biotechniques
2. Maniatis, T., E.T. Fritsch and J. Sambrook, 1982.
Molecular Cloning: A Laboratory Manual.
3. Winberg. G., 1991. A
Rapid Method for Preparing DNA from Blood. Suited for PCR Screening
of Transgenes in Mice. PCR Methods Applic. 1:72-74.