Bio-Rad RNA Mini Kit User Manual

Aurum

™

Total RNA Mini Kit

Instruction Manual

Catalog # 732-6820

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

Section 2 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .1

Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .2

Section 4 Necessary Supplies . . . . . . . . . . . . . . . . . . . . . .2

Section 5 Before Using the

Aurum™ Total RNA Mini Kit . . . . . . . . . . . . . . . . . .3

Starting Material Amounts . . . . . . . . . . . . . . . . . . . . . . .3

Reagents Used With the Aurum Total RNA Mini Kit . . . .5

Elution Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Ribonucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Disruption and Homogenization . . . . . . . . . . . . . . . . . . .6

Section 6 Vacuum Manifold Setup and Use With the

Column Adaptor Plate (CAP) . . . . . . . . . . . . . . .8

Guidelines for Vacuum format . . . . . . . . . . . . . . . . . . . .8

About the CAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Preparing the Aurum Vacuum Manifold . . . . . . . . . . . . .9

Vacuum Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Manifold Wash Setup . . . . . . . . . . . . . . . . . . . . . . . . . .10

Section 7 Vacuum Protocol . . . . . . . . . . . . . . . . . . . . . . .11

Section 8 Spin Protocol . . . . . . . . . . . . . . . . . . . . . . . . . .15

Section 9 Troubleshooting Guide . . . . . . . . . . . . . . . . . . .19

Section 10 Ordering Information . . . . . . . . . . . . . . . . . . . .22

Section 1
Introduction

The Aurum™ total RNA mini kit purifies total RNA samples rapidly from
mammalian cell cultures, bacteria, and yeast (Saccharomyces cerevisiae), as
well as animal and plant tissue. Total RNA samples prepared using the Aurum
total RNA mini kit are suitable for use in a variety of downstream applications,
including reverse transcription-PCR (RT-PCR), real-time PCR, and northern
blots. A DNase l digest during the purification effectively removes genomic
DNA contamination from the preparation, eliminating the need for separate
DNase digests. All solutions and binding columns in the kit are RNase-free,
ensuring the integrity of the isolated total RNA. The Aurum total RNA mini kit
may be used in a spin or vacuum format using the Aurum vacuum manifold
(catalog # 732-6470).

Section 2
Kit Components

The Aurum™ total RNA mini kit contains the following components:

Components* Quantity/Amount

RNA binding columns 50
Capless wash tubes, 2 ml 50
Capped microcentrifuge tubes, 1.5 ml 50
Capped microcentrifuge tubes, 2.0 ml 100
DNase I (lyophilized) 1 vial

Lysis solution 50 ml
Low stringency wash solution
(5x concentrate) 20 ml
High stringency wash solution 40 ml
Elution solution 20 ml
DNase dilution solution 20 ml

*There may be reagent remaining in some bottles.

1

Section 3
Storage Conditions

All kit components (including lyophilized DNase I) should be stored at room
temperature. Store reconstituted DNase I at –20°C in a nonfrost-free freezer,
avoiding repeated freeze-thaw cycles. If precipitation is observed in any
solution, warm the solution to 37°C to redissolve and allow the solution to
return to room temperature before use.

Section 4
Necessary Supplies

Equipment and reagents to be provided by the customer:

• Microcentrifuge (>12,000 x g)

• b-mercaptoethanol, 14.2 M (catalog # 161-0710)

• Lyticase (for yeast RNA isolation only)

• Lysozyme (for bacterial RNA isolation only)

• Isopropanol (for bacterial RNA isolation only)

• Polyvinylpyrrolidone-40 (PVP), 2% (for plant isolation only)

• 95–100% ethanol

• Tris for DNase I reconstitution (catalog # 161-0716)

Additional equipment required for vacuum format:

• Aurum™ vacuum manifold with vacuum regulator and column adaptor plate
(catalog # 732-6470), or other vacuum manifold with luer fittings

• Vacuum source (capability of –23 inHg required)

2

Section 5
Before Using the
Aurum Total RNA Mini Kit

Please read the following guidelines before proceeding with the total RNA
purification.

Starting Material Amounts

The Aurum total RNA mini kit is designed to process up to the amounts
indicated below (per column):

•2 x 10

6

mammalian cultured cells

•3 OD

•ml* of gram-positive or gram-negative bacteria

3 OD

•ml of bacteria roughly corresponds to a culture volume of 500–750 µl

•3 OD

•ml of yeast (S. cerevisiae)

3 OD

•ml of yeast roughly corresponds to a culture volume of 600–1,000 µl

• 40 mg animal tissue

• 60 mg plant tissue

Processing larger amounts of starting material may lead to column clogging
and reduced RNA purity.

*Spectrophotometric determination of bacterial or yeast culture density is a
REQUIREMENT for optimal total RNA isolation from these starting materials.
To determine the density of a bacterial or yeast culture (OD

600

), combine 50 µl

of culture with 950 µl growth medium (20-fold dilution). Use the growth
medium as a blank and take the spectrophotometric reading at l = 600 nm.
Multiply this figure by 20 to calculate the OD

600

value of the undiluted bacterial

or yeast culture. Depending upon the OD

600

value, a specific volume of the
culture will be selected to provide an optimum amount of bacteria or yeast for
processing. To calculate the volume of culture required, use the following
equation:

(OD

600

of undiluted culture)** x (culture volume in ml) = # OD•ml

For example, 3 OD•ml of yeast would require 500 µl of an undiluted culture
with an OD

600

= 6.

** 1 OD

600

is equivalent to approximately 8 x 108bacterial cells/ml, or 1 x 10

7

yeast cells/ml.

3

Table 1. Yield (per column) of total RNA from various samples
using the Aurum total RNA mini kit.

Starting Material Avg. Yield (µg)*

Cultured cells (2 x 106)

3T3 20–30
HeLa 20–40

Bacteria (2.4 x 10

9

)

E. coli 30–35
B. cereus 30–35

Yeast (3 x 10

7

)

S. cerevisiae 20–25

Animal tissue (up to 40 mg)

Brain 15–20
Liver 8–12
Lung 8–12
Kidney 15–20
Spleen 15–25

Plant tissue (up to 60 mg)

Arabidopsis 5–10
Maize 5–10
Potato 15–20
Spinach 10–15
Tomato 5–10
Tobacco 5–10

Starting material amounts in parentheses are the maximum amounts recommended for use with the
total RNA mini kit.

*Yield figures are representative of a minimum of 20 mini column preps performed in both
vacuum and spin formats.

4

5

Reagents Used With the Aurum Total RNA Mini Kit

• The low stringency wash solution is provided as a 5x concentrate. Add
4 volumes (80 ml) of 95–100% ethanol to the low stringency
wash solution concentrate before initial use

• Before using the lysis solution, add 500 µl of b-mercaptoethanol
(catalog # 161-0710) to the solution for a final concentration of 1%

• The RNase-free DNase I is provided as a lyophilized powder. Reconstitute
the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not supplied) to the vial
and pipetting up and down briefly to mix. Do not vortex. Store the
reconstituted DNase I at –20°C in a nonfrost-free freezer

• Bacterial total RNA isolations require the use of TE (10 mM Tris,
1 mM EDTA, pH 7.5), which is not supplied with the kit

• Yeast total RNA isolations require the use of lyticase dilution buffer
(1 M sorbitol, 0.1 M EDTA, pH 7.4, 0.1% b-mercaptoethanol), which is
not supplied with the kit

• Vendors of lyticase, which is used to partially degrade the cell walls of yeast
cells, may have different definitions of the enzyme’s activity. As used in this
instruction manual, 1 unit of lyticase produces a DA

800

of 0.001/min at
pH 7.5 at 25°C, using 3 ml of yeast suspension as a substrate in a 3 ml
reaction volume