Bio-Rad Bio-Spin 6 and Micro Bio-Spin 6 Columns User Manual

Introduction

Micro Bio-Spin chromatography columns are ready to use for
rapid and efficient cleanup and purification of nucleic acids
and proteins using a microcentrifuge.

Micro Bio-Spin Columns

• Remove dye terminators

• Remove unincorporated nucleotides

• Desalt nucleic acids, proteins and peptides
The columns are packed with special grades of Bio-Gel®P poly-

acrylamide P-6 or P-30 gel matrices manufactured specifically
for Bio-Rad spin columns. This unique gel produces very effi­cient, non-interactive size separations. Micro Bio-Spin columns
are suitable for use with 1.5 or 2.0 ml microcentrifuge tubes and
are completely autoclavable.

T echnical Information

Gel Matrix

Bio-Gel P-6 or P-30 polyacrylamide gel suspended in 1.0 ml
of buffer

Buffers

SSC buffer (150 mM sodium chloride, 17.5 mM sodium cit­rate, pH 7.0) with 0.02% sodium azide

Tris buffer (10 mM Tris-HCl, pH 7.4) with 0.02% sodium
azide

Sample Application Volumes

Nucleic acids, proteins, and peptides, 20–75 µl. Volumes less
than 20 µl may affect recovery

Exclusion Limits

Bio-Gel P-6 gel: 5 base pairs (nucleic acids) or 6,000 daltons
(proteins,peptides)

Bio-Gel P-30 gel: 20 base pairs (nucleic acids) or 40,000 dal­tons (proteins, peptides)

Expected Retention and Recovery

Micro Bio-Spin 6 column
98% retention of unincorporated nucleotides at 20 µl

90% recovery of applied DNA at 20 µl

Micro Bio-Spin 30 column
100% retention of unincorporated nucleotides at 20 µl

95% recovery of applied DNA at 70 µl

Centrifuge Type

Microcentrifuge with a centrifugal force of 1,000 x (g).

Autoclavability

Micro Bio-Spin columns, Bio-Gel P gel, and collection tubes are
completely autoclavable at 121 °C for 30 minutes at pH 6.0–8.0.

Chemical Stability

pH 2–10, common aqueous buffers, formamide, dilute organic
acids, alcohol, 20% (V/V) other chaotropic agents, detergents.

Storage

Store at 4 °C. Do not freeze. Shelf life is 1 year at 4 °C.

Instructions for Use

1. Invert the column sharply several times to resuspend the
settled gel and remove any bubbles. Snap off the tip and
place the column in a 2.0 ml microcentrifuge tube
(included). Now remove the top cap. If the column does
not begin to flow, push the cap back on the column and
then remove it again to start the flow. Allow the excess
packing buffer to drain by gravity to the top of the gel bed
(about two minutes). Discard the drained buffer then
place the column back into the 2.0 ml tube.

2. Centrifuge for 2 minutes in a microcentrifuge at 1,000 x (g)
(see Centrifugation Notes section) to remove the remain­ing packing buffer. Discard the buffer.

3. Place the column in a clean 1.5 or 2.0 ml microcentrifuge
tube. Carefully apply the sample (20–75 µl) directly to the
center of the column. Application of more or less than the
recommended sample volume may decrease column per­formance.

4. After loading sample, centrifuge the column for 4 minutes
at 1,000 x (g).

5. Following centrifugation, the purified sample is now in
either SSC or Tris buffer. Molecules smaller than the col­umn’s exclusion limit will be retained by the column (see
Specifications).

6. Properly dispose of the used column.

Micro Bio-Spin®Chromatography Columns

Buffer Exchange

The gel in the Micro Bio-Spin columns is suspended in either
SSC buffer, pH 7.0, or Tris buffer, pH 7.4. The gel matrix is
compatible with most aqueous buffers. Buffer exchange can be
achieved using the following procedure.

1. Follow steps 1 and 2 in the Instructions for Use section.

2. Apply the new buffer in 500 µl aliquots. After each appli­cation of new buffer, let the buffer drain out by gravity, or
centrifuge the column for 1 minute to remove the buffer.
Discard buffer from collection tube. Repeat as required.
Three washes result in > 99% of the buffer exchanged. Four
washes result in > 99.9% of buffer exchanged.

3. Sample can now be applied to the column as directed in
steps 3 through 6 in the Instructions for Use section.

Centrifugation Notes

Micro Bio-Spin columns fit 1.5 ml microcentrifuge tubes for
sample collection during centrifugation. Use the 2.0 ml micro­tubes provided with the columns for the initial column equili­bration step.

Benchtop microcentrifuges capable of generating a minimum
force of 1,000 x (g) are suitable for Micro Bio-Spin column use.
The gravitational force created at a particular rpm is a function
of the radius of the microcentrifuge rotor. Consult the micro­centrifuge instruction manual for conversion information from
rpm to g-force. Alternatively, to calculate the speed (rpm)
required to reach a gravitational force of 1,000 x (g), use the
following equation:

RCF (g) = (1.12 x 10

-5

)(rpm)2r

where r is the radius in centimeters measured from the center
of the rotor to the middle of the Micro Bio-Spin column, and
rpm is the speed of the rotor in revolutions per minute.

Sample Dilution T roubleshooting Guide

Sample dilution will occur if, during column equilibration, the
column is centrifuged without first removing the excess pack­ing buffer. Dilution will also occur if the excess packing buffer
is left in the collection tube prior to centrifugation. The tip of
the column becomes submerged and retains excess buffer.

To correct this situation, always discard the buffer from the
collection tube after gravity draining and after the first cen­trifugation (steps 1 and 2 of Instructions for Use section).

Sterilization

If a sterile Micro Bio-Spin column is required, autoclave the
column at 121 °C for 20–30 minutes. If exchanging buffers,
the buffer pH in the column should be in the range of 6.0 to

8.0 prior to autoclaving.

4006051 Rev B

Ordering Information

Catalog
Number Product Description

Bio-Gel P-6 Columns in SSC

732-6200 Micro Bio-Spin 6 Column, SSC, 25, includes

25 P-6 columns in SSC buffer, 25 buffer col­lection tubes, and instruction manual

732-6201 Micro Bio-Spin 6 Column, SSC, 100, includes

100 P-6 columns in SSC buffer, 100 buffer col­lection tubes, and instruction manual

732-6205 Micro Bio-Spin 6 Column, SSC, 1,000,

includes 1,000 P-6 columns in SSC buffer, 1,000
buffer collection tubes, and instruction manual

Bio-Gel P-6 Columns in Tris

732-6221 Micro Bio-Spin 6 Column, Tris, 25, includes

25 P-6 columns in Tris buffer, 25 buffer collec­tion tubes, and instruction manual

732-6222 Micro Bio-Spin 6 Column, Tris, 100, includes

100 P-6 columns in Tris buffer, 100 buffer col­lection tubes, and instruction manual

732-6225 Micro Bio-Spin 6 Column, Tris, 1,000, includes

1,000 P-6 columns in Tris buffer, 1,000 buffer
collection tubes, and instruction manual

Bio-Gel P-30 Columns in SSC

732-6202 Micro Bio-Spin 30 Column, SSC, 25, includes

25 P-30 columns in SSC buffer, 25 buffer col­lection tubes, and instruction manual

732-6203 Micro Bio-Spin 30 Column, SSC, 100,

includes 100 P-30 columns in SSC buffer, 100
buffer collection tubes, and instruction manual

732-6206 Micro Bio-Spin 30 Column, SSC, 1,000,

includes 1,000 P-30 columns in SSC buffer, 1,000
buffer collection tubes, and instruction manual

Bio-Gel P-30 Columns in Tris

732-6223 Micro Bio-Spin 30 Column, Tris, 25, includes

25 P-30 columns in Tris buffer, 25 buffer col­lection tubes, and instruction manual

732-6224 Micro Bio-Spin 30 Column, Tris, 100,

includes 100 P-30 columns in Tris buffer, 100
buffer collection tubes, and instruction manual

732-6226 Micro Bio-Spin 30 Column, Tris, 1,000, includes

1,000 P-30 columns in Tris buffer, 1,000 buffer
collection tubes, and instruction manual

Empty Columns

732-6204 Empty Micro Bio-Spin Column, 100,

includes 100 empty columns, 100 buffer col­lection tubes, and instruction manual

Life Science
Group

Bio-Rad
Laboratories

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