Bio-Rad PureZOL RNA Isolation Reagent User Manual
PureZOL™RNA Isolation
Reagent
Instruction Manual
Catalog #732-6890
For technical support,
call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD
(1-800-424-6723)
Table of Contents
Section 1 Introduction...........................................................1
Kit Components.................................................................2
Storage and Stability.........................................................2
Special Handling Instructions............................................2
Section 2 Materials and Equipment Required
(Not Provided).......................................................3
Section 3 Maintaining an RNase-Free Environment...........4
Preparing Reagents Not Supplied with the Kit..................5
Section 4 Recommendations for Best Results...................6
Section 5 Protocol..................................................................7
Section 6 Troubleshooting..................................................12
Section 7 Reference.............................................................16
Section 8 Ordering Information..........................................17
Section 1
Introduction
PureZOL RNA isolation reagent is intended for the extraction of
total RNA from animal and plant tissues, cultured mammalian
cells, and bacterial and yeast cells in under 1 hour.PureZOL can
also be used for the simultaneous extraction of RNA, DNA, and
proteins from various samples.This reagent allows processing of
small amounts of starting material (50 cells or 5 mg of tissue), and
can be scaled up to process large amounts of starting material.
PureZOL RNA isolation reagent is a monophasic solution of
phenol and guanidine isothiocynate, an improved version of the
single-step isolation of total RNA developed by Chomczynski and
Sacchi (1987).The reagent facilitates immediate and effective
inhibition of RNase activity, while lysing cells and eliminating other
cellular components. Following the addition of chlorofor m and
subsequent centrifugation, the homogenate separates into an
aqueous phase, an interphase and an organic phase. RNA is
recovered in the aqueous phase with the addition of isopropyl
alcohol, and is subsequently washed in ethanol and solubilized in
RNase-free water.
Total RNA isolated from this procedure is generally DNA- and
protein-free and can be used for northern blot analysis,
in vitro
translation, poly (A)+selection, RNase protection assays, RT-PCR,
and molecular cloning. For RT-PCR analysis, DNase treatment may
be necessary for optimal results. Subsequent cleanup of RNA using
an Aurum™ total RNA mini kit (732-6820) is recommended to
remove any phenol and other contaminants that may have
coprecipitated with the RNA (see section 8 for ordering information).
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Kit Components
Catalog # 732-6890 – PureZOL RNA isolation reagent, 100 ml
Storage and Stability
PureZOL RNA isolation reagent is shipped at room temperature.
This product is guaranteed for 12 months from the date of
purchase when stored at 2–8°C and kept away from light.
Special Handling Instructions
PureZOL RNA isolation reagent contains a poison (phenol) and an
irritant (guanidine thiocynate).The reagent causes burns and can
be fatal if ingested.When working with PureZOL, use gloves and
eye protection (laboratory glasses, shield, and safety goggles).Do
not get on skin or clothing. Avoid breathing vapor. Read warning
notice on bottle and MSDS.
In case of contact:Immediately flush eyes or skin with a large
amount of water for at least 15 minutes and seek immediate
medical attention.
2
Section 2
Materials and Equipment Required
(Not Provided)
Microcentrifuges (capable of >12,000 x g) — at 4°C and at room
temperature
95–100% ethanol, ACS grade or better
Chloroform (without additives such as isoamyl alcohol);need
0.2 ml of chloroform per 1 ml of PureZOL
Isopropyl alcohol; need 0.5 ml per 1 ml of PureZOL
RNase-free water (DEPC-treated water)
75% ethanol (prepared in DEPC-treated water); need 1 ml per
1 ml of PureZOL
RNase-free pipet tips and micropipets
RNase-free polypropylene centrifuge tubes with caps, capable of
withstanding centrifugal forces of 12,000 x g
Disposable latex or vinyl gloves
Supplies for tissue grinding, disruption, and homogenization
• Fresh tissue — tissue cutter and a ruler to measure size
• Frozen tissue — liquid nitrogen and mortar and pestle
• Tissue homogenizer — dounce homogenizers, rotor-stator
homogenizers, or bead mill homogenizers are recommended
Optional — Aurum total RNA mini kit (732-6820) for RNA cleanup
following isolation of RNA using PureZOL (see section 8 for ordering information)
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Section 3
Maintaining an RNase-Free Environment
To avoid introducing RNases, great care must be taken in
handling the reagents and purified RNA samples. Care must be
taken to proceed through the RNA isolation as quickly as possible.
An RNase-free environment will yield the best results.
If possible, work in an RNase-free environment;use latex or vinyl
gloves when handling reagents or RNA and change gloves
frequently.
Nondisposable, nonautoclavable plasticware should be rinsed with
0.1 M NaOH, 1 mM EDTA followed by several rinses with diethyl
pyrocarbonate (DEPC)-treated water before use. DEPC is an
efficient, strong, and nonspecific RNase inhibitor that is usually
used at a concentration of 0.1%.
Warning:DEPC is suspected to
be a carcinogen and should be handled with care
.
Always use
gloves and open under a fume hood
.
Glassware and other autoclavable items may be treated using the
DEPC method described above for nonautoclavable plasticware, or
by baking for 4 hours at 300°C.
Working surfaces and pipets should be kept clean and wiped
periodically with a 0.5 M NaOH solution.
Use sterile plasticware and machine-packaged aerosol-resistant
pipet tips. Pour tubes from an unopened bag (or bag marked "For
RNA Use Only") onto an RNase-free environment (such as plastic
wrap). Otherwise, sterilize by autoclaving.
4
Solutions that are prepared by the user should be treated with
DEPC to inactivate RNases.See instructions below on how to
prepare DEPC-treated solutions.
Keep reagent bottles closed when not in use and keep RNA
samples on ice to prevent degradation by RNases.
Preparing Reagents Not Supplied With the Kit
Note: DEPC is destroyed by primary amines. If a solution
containing a primary amine will be DEPC-treated, omit the amine
in preparing the solution. Perform the DEPC treatment as
described above, and add the amine to the autoclaved solution
once the solution has cooled.
Preparation of DEPC-treated water
.To prepare a 0.1% (v/v)
solution of DEPC-treated water, add 1.0 ml of liquid DEPC per
1 L of water.Incubate the solution at 37°C for 1 hour while mixing
thoroughly. Autoclave the treated water to remove the DEPC.
Preparation of 75% ethanol
.To prepare 100 ml of 75% ethanol,
add 75 ml of 95–100% ethanol to 25 ml of DEPC-treated water.
Mix well before use.
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