Table of Contents

BIO BASIC INC. Introduction

EZ-10 SPIN COLUMN HANDBOOK 1. Limitations of Use p3

2. Features p3
3. Applications p4
EZ-10 Spin Column Plasmid DNA MiniPreps Kit
EZ-10 Spin Column PCR Products Purification Kit Storage p4
EZ-10 Spin Column DNA Gel Extraction Kit
Quality Control p4

EZ-10 Spin Column Plasmid DNA Minipreps Kit

1. Protocol p5
2. Low copy plasmid protocol p8
3. Troubleshooting guide p9

EZ-10 Spin Column PCR Products Purification

Kit
Version 3.0 1. Protocol p11
Rev 9/24/2011 2. Troubleshooting guide p18
ISO9001 Certified
EZ-10 Spin Column DNA Gel Extraction Kit
1. Protocol p13
2. Troubleshooting guide p18

20 Konrad Crescent, Markham Ontario L3R 8T4 Canada Other Kits Available p19
Tel: (905) 474 4493, (800) 313 7224 Fax: (905) 474 5794
Email: [email protected] Web: www.biobasic.com

EZ-10 Spin Column Handbook 2

Introduction Applications
The EZ-10 Spin Column Kits provide a simple and
efficient method for purification of plasmid DNA, EZ-10 Spin Column Plasmid DNA Minipreps Kit
extraction of DNA from agarose gels, and BS413, BS414, BS614,
purification of DNA from enzymatic reactions such This kit can be used for purification of plasmid DNA
as PCR or restriction enzyme digestions. from 40bp-40kb

The DNA is selectively adsorbed in silica gel-based EZ-10 Spin Column PCR Products Purification
EZ-10 column and other components are washed Kit BS363, BS364, BS664
away. The DNA is then eluted off the column and Recovery of 40bp-40kb DNA fragments from
can be used for any downstream applications. reaction solutions

The purification method used in these protocols EZ-10 Spin Column DNA Gel Extraction Kit
does not require use of phenol, chloroform, or CsCl. BS353, BS354, BS654
The DNA is purified without an additional step of Recovery of 40bp-40kb DNA fragments from
ethanol precipitation. agarose gels

Limitations of Use Storage

These kits are designed for research use only. The EZ-10 Spin Column Kits should be stored dry at
purified plasmid DNA should not be used for live room temperature (15oC-25oC). Kits can be stored
animal transfections. It is also not to be used for for up to 24 months without showing any reduction
human diagnostic or drug production purposes. in performance and quality. RNase A stock solution
can be stored for 2 years at 4oC. After addition of
Features RNase A, Solution I is stable for 6 months at 4oC.
√ Simple, Fast and Efficient
√ Preparation of high quality DNA which can be Quality Control
used in any downstream applications such as Each lot of EZ-10 Spin Column kit is tested against
sequencing, PCR, transformation or restriction predetermined specifications to ensure consistent
digestions product quality.
√ High Yield and Reproducible
√ High Capacity - Up to 10µg of DNA per column

EZ-10 Spin Column Handbook 3 EZ-10 Spin Column Handbook 4

 d) Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5.
Protocol: EZ-10 Spin Column Plasmid DNA Minipreps Although TE buffer pH 8.0 or water may be
Kit BS413, BS414, BS614 substituted, the resulting yields may be up to
20% lower.
EZ-10 Spin Column BS413 BS414 BS614
Plasmid DNA 50 100 250 Principle:
Minipreps Kit Preps Preps Preps This kit provides a simple and efficient method for
Components mini plasmid DNA purification. The plasmid DNA is
RNase A Solution 120µl 240µl 600µl selectively adsorbed in silica gel-based EZ-10
(10mg/ml) column and other impurities such as proteins, salts,
Solution I 6ml 12ml 30ml nucleotides, oligos (<40-mer) are washed away.
Solution II 12ml 24ml 2X30ml The plasmid DNA is then eluted off the column and
Solution III 25ml 2X25ml 5X25ml can be used for any downstream application.
Wash Solution 20ml 2X20ml 2X40ml
Elution Buffer 5ml 10ml 25ml Protocol for Purification of Plasmid DNA
EZ-10 Column 50 100 250 1. Add 1.5 - 5mL overnight culture in the tube and
Protocol 1 1 1 centrifuge at 12,000rpm for 2 minutes. Drain the
liquid completely. For low copy number
a) Before use, add the RNase A Solution to the plasmid, see the protocol on the following page.
bottle containing Solution I and mix well. 2. Add 100µl of Solution I to the pellet, mix well,
Solution I with RNase A should be stored at 4ºC and keep for 1 minute.
for frequent use and at -20ºC for infrequent use. 3. Add 200µl of Solution II to the mixture, and
b) Solution II may form a precipitate upon storage. mix gently by inverting the tube 4-6 times and
If necessary, dissolve the precipitate by then keep at room temperature for 1 minute. To
warming the solution at 37ºC. prevent contamination from genomic DNA, do
c) Before use, add 80ml of 96-100% of ethanol to not vortex.
20ml Wash Solution for BS413; add 160ml of 4. Add 350µl of Solution III, and mix gently.
96-100% ethanol to 40ml Wash Solution for Incubate at room temperature for 1 minute.
BS414; add 320ml of 96-100% ethanol to 80ml 5. Centrifuge at 12,000rpm for 5 minutes.
Wash Solution for BS614. For other volumes of 6. Transfer the above supernatant (step 5) to the
wash solution, simply add enough ethanol to EZ-10 column. Centrifuge at 10,000rpm for 2
make a 4:1 ratio (volume of added ethanol: minutes.
volume of Wash Solution = 4:1).

EZ-10 Spin Column Handbook 5 EZ-10 Spin Column Handbook 6

7. Discard the flow-through in the tube. Add 750µl EZ-10 Spin Column BS4139 BS4149 BS6149
of Wash Solution to the column, and centrifuge Plasmid DNA 50 100 250
at 10,000rpm for 2 minutes. Minipreps Kit Preps Preps Preps
Components
RNase A Solution 240µl 480µl 1.2ml
8. Repeat wash procedure in step 7. (10mg/ml)
9. Discard the flow-through in the collection tube. Solution I 12ml 24ml 60ml
Centrifuge at 10,000rpm for an additional Solution II 24ml 2X24ml 4X30ml
minute to remove any residual Wash Solution. Solution III 2X20ml 3X25ml 6X30ml
10. Transfer the column to a clean 1.5ml microfuge Wash Solution 20ml 2X20ml 2X40ml
tube. Add 50µl of Elution Buffer into the center Elution Buffer 5ml 10ml 25ml
part of the column and incubate at room EZ-10 Column 50 100 250
temperature for 2 minutes. Centrifuge at 10,000 Protocol 1 1 1
rpm for 2 minutes.
11. Store purified DNA at -20ºC.

Note: It is extremely important to add the Elution 1. Use 5 - 10ml overnight culture. Add overnight
Buffer into the center part of the column. Incubating culture to a 1.5ml microfuge tube and centrifuge
the column with the Elution Buffer at higher at 12,000rpm for 2 minutes. Drain the liquid
temperature (37ºC to 50ºC) may slightly increase completely and repeat with another portion of
the yield especially for large (>10,000bp) DNA culture (in the same tube).
2. Add 200µl of Solution I to the pellet, mix well
Plasmids. Prewarming the Elution Buffer at 55ºC to
80ºC may also slightly increase elution efficiency. and keep for 1 minute.
3. Add 400µl of Solution II to the mixture, and
mix gently by inverting the tube 4-6 times and
Protocol for Purification of Low Copy Plasmid then keep at room temperature for 1 minute. To
DNA prevent contamination from genomic DNA, do
not vortex.
4. Add 700µl of Solution III, and mix gently.
Incubate at room temperature for 1minute.
5. Centrifuge at 12,000 rpm for 5 minutes.
6. Transfer half of the above supernatant (step 5)
to the EZ-10 column. Let the column stand for 2
minutes. Centrifuge at 10,000 rpm for 2 minutes.

EZ-10 Spin Column Handbook 7 EZ-10 Spin Column Handbook 8

 Discard the flow-through in the tube and add shaking speed provides sufficient aeration
the second half of the supernatant, centrifuge of the culture.
again at 10,000 rpm for 2 minutes.
d. Very high cell density, therefore incomplete
7. Discard the flow-through in the tube. Add 750µl
cell lysis. Double the volume of Solution I, II
of Wash Solution to the column, and centrifuge
and III.
at 10,000rpm for 2 minutes.
8. Repeat wash procedure in step 7. 2) Contamination of chromosomal DNA
9. Discard the flow-through in the collection tube. Do not vortex the sample after adding solution II
Centrifuge at 10,000 rpm for an additional and III. Vigorous shaking will cause shearing of
minute to remove any residual Wash Solution. chromosomal DNA. Smaller pieces of
10. Transfer the column to a clean 1.5ml microfuge chromosomal DNA will be captured on silica gel
tube. Add 50µl of Elution Buffer into the center and carried over with purified plasmid DNA.
part of the column and incubate at room
3) RNA contamination
temperature for 2 minutes. Centrifuge at 10,000
RNase activity is weakened or lost. Add
rpm for 2 minutes.
addition RNase A to Solution I and store at 4oC.
11. Store purified DNA at –20ºC.
Note: If using Low Copy Plasmid protocol, extra 4) OD 260nm/280nm ratio outside 1.9-2.2 range
solutions are required and these solutions are also If the ratio of OD260nm/280nm is greater than
available for purchase separately from Bio Basic 2.2, there may be traces of ethanol present.
Inc.
If the ratio of OD260nm/280nm is smaller than
Troubleshooting Guide: EZ-10 Spin Column
1.9, there is chance of protein contamination.
Plasmid DNA Minipreps Kit
Make sure the sample is mixed well after
1) Low Yield Solution III is added and after spinning down.
There are a number of variables that can cause
5) For optimal results in downstream DNA
low yield:
sequencing, an additional washing step is
a. Each of the steps has to be strictly followed.
recommended.
b. Make sure there is no precipitant in Solution
I, II or III. If precipitant is present in the
buffer, warm up the solution to 37oC and
shake well.
c. Low culture density. Make sure that the
temperature in the incubator is stable and

EZ-10 Spin Column Handbook 9 EZ-10 Spin Column Handbook 10

 bind to the membrane and are washed away. The
DNA fragments can then be eluted off the column
Protocol: EZ-10 Spin Column PCR Products
in small volume and used in downstream
Purification Kit BS363, BS364, BS664
applications without further processing.
EZ-10 Spin Column BS363 BS364 BS664
PCR 50 100 250
Purification Kit Preps Preps Preps Protocol for Purification of PCR Products
Components 1. Transfer PCR reaction mixture to a 1.5ml
Binding Buffer I (New) 20ml 2x20ml 4x25ml microfuge tube and add 3 volumes of Binding
Wash Solution (A) 20ml 2X20ml 2X40ml Buffer I.
Elution Buffer(B) 5ml 10ml 25ml 2. Transfer the above mixture solution to the EZ-10
EZ-10 Column 50 100 250 column and let it stand at room temperature for 2
Protocol 1 1 1 minutes. Centrifuge at 10,000 rpm for 2 minutes.
3. Remove the flow-through in the tube. Add 750µl
(A) Before use, add 80ml of 96-100% of ethanol to of Wash Solution to the column and centrifuge at
20ml Wash Solution for BS363; add 160ml of 96- 10,000 rpm for 2 minutes.
100% ethanol to 40ml Wash Solution for BS364; 4. Repeat washing procedure in step 3. Spin at
add 320ml of 96-100% ethanol to 80ml Wash 10,000 rpm for an additional minute to remove
Solution for BS664. For other volumes of Wash any residual Wash Solution.
Solution, simply add enough ethanol to make a 5. Transfer the column into a clean 1.5ml microfuge
4:1 ratio (volume of added ethanol: volume of tube and add 30-50µl of Elution Buffer. Incubate
Wash Solution = 4:1). at room temperature for 2 minutes. Centrifuge at
(B) Elution Buffer is 2mM Tris-HCl pH 8.0~8.5. 10,000 rpm for 2 minutes to elute the DNA.
Although TE buffer pH 8.0 or water may be
substituted, the resulting yields may be up to 20% Note: It is extremely important to add the Elution
lower. Buffer into the center part of the column. Incubating
the column with the Elution Buffer at higher
Principle: temperature (37ºC to 50ºC) may slightly increase the
EZ-10 spin column purification kits utilize a silica- yield especially of large (>10,000 bp) DNA plasmids.
gel membrane that selectively absorbs up to 10µg Prewarming the Elution Buffer at 55ºC to 80ºC may
of DNA fragments in the presence of specialized also slightly increase elution efficiency.
binding buffers. Nucleotides, oligos (<40-mer),
enzymes, mineral oil and other impurities do not 6. Store purified DNA at -20ºC.

EZ-10 Spin Column Handbook 11 EZ-10 Spin Column Handbook 12

 (B) Elution Buffer is 2mM Tris-HCl pH 8.0~8.5.
Although TE buffer pH 8.0 or water may be
Note: substituted, the resulting yields may be up to
1. If PCR reaction mixture contains seriously non- 20% lower.
specific amplified DNA fragments, use of the
DNA Gel Extraction Kit is recommended. Note: The kit is observed to have better
2. This kit can not remove the template and performance when TAE, rather than TBE, is used.
primers with chain length longer than 40-mer.
Principle:
The EZ-10 spin column purification kit utilizes a
Protocol: EZ-10 Spin Column DNA Gel silica-gel based membrane which selectively
Extraction Kit BS353, BS354, BS654 adsorbs up to 10µg of DNA fragments in the
presence of specialized binding buffers.
EZ-10 Spin BS353 BS354 BS654 Nucleotides, oligos (<40- mer), enzymes, mineral
Column DNA 50 100 250 oil and other impurities do not bind to the
Gel Extraction Preps Preps Preps membrane and are washed away. DNA fragments
Kit Components are then eluted off the column and can be used for
Binding Buffer II 50ml 2X50ml 5X50ml downstream applications without further processing.
Wash Solution 20ml 2X20ml 2X40ml
Elution Buffer 5ml 10ml 25ml Protocol for Agarose Gel
EZ-10 Column 50 100 250
Protocol 1 1 1 1. Excise the DNA fragment from the gel with a
clean, sharp scalpel. Weigh the gel slice and
(A) Before use, add 80ml of 96-100% of ethanol to transfer to a 1.5mL microfuge tube.
20ml Wash Solution for BS353; add 160ml of 96- 2. Add 400µl of Binding Buffer II for each 100mg
100% ethanol to 40ml Wash Solution for BS354; of gel weight (for example, a gel slice weighing
add 320ml of 96-100% ethanol to 80ml Wash 125mg would require 500µl of Binding Buffer II).
Solution for BS654. For other volumes of wash Incubate at 500C-600C for 10 minutes and
solution, simply add enough ethanol to make a 4:1 shake occasionally until agarose is completely
ratio (volume of added ethanol: volume of Wash dissolved. For high concentration gels (1.5-
Solution = 4:1). 2.0%), 700µl of Binding Buffer II per 100mg of
agarose gel are added.

EZ-10 Spin Column Handbook 13

EZ-10 Spin Column Handbook 14
Note: After addition of binding buffer, carefully Protocol for DNA purification from enzymatic
monitor the color of the binding mixture. If the reactions
binding mixture is yellow, then optimal pH has 1. Transfer entire contents of the reaction mixture
been obtained; continue with the rest of extraction to a 1.5ml microfuge tube and add 3 volumes
steps. However, if the binding mixture turns a blue of Binding Buffer II. Mix by inverting the tube a
or purple color, adjust pH by adding a small volume few times.
of 3 M sodium acetate (pH 5.0) until optimal pH is 2. Add the above mixture to the EZ-10 column and
reached. Proceed with the rest of the extraction let the column stand for 2 minutes. Centrifuge
steps. at 10,000rpm for 1 minute and discard the flow-
through in the tube.
3. Add the above mixture to the EZ-10 column and 3. Add 750µl of Wash Solution, and centrifuge at
let stand for 2 minutes. Centrifuge at 10,000rpm 10,000rmp for 1 minute. Discard the solution
for 2 minutes and discard the flow-through in in the tube.
the tube. 4. Repeat step 3. Spin at 10,000rpm for an
4. Add 750µl of Wash Solution, and centrifuge at additional minute to remove any residual Wash
10,000rpm for one minute. Discard the solution Buffer.
in the tube. 5. Place the column in a clean 1.5ml microfuge
5. Repeat step 4. Centrifuge at 10,000rpm for an tube. Add 30-50µl of Elution Buffer to the center
additional minute to remove any residual Wash of the column and incubate at room
Buffer. temperature for 2 minutes.
6. Place the column in a clean 1.5ml microfuge
tube. Add 30-50µl of Elution Buffer to the center Note: It is extremely important to add the Elution
of the column and incubate at room Buffer into the center part of the column. Incubating
temperature for 2 minutes. Centrifuge at the column with the Elution Buffer at higher
10,000rpm for 2 minutes to elute DNA. temperature (37ºC to 50ºC) may slightly increase
the yield especially for large (>10,000bp) DNA
Note: It is extremely important to add the Elution Plasmids. Prewarming the Elution Buffer at 55ºC to
Buffer to the center of the column. Incubating the 80ºC may also slightly increase elution efficiency.
column at higher temperature (37ºC to 50ºC) may
slightly increase the yield. Pre-warming the Elution 6. Centrifuge at 10,000rpm for 2 minutes to elute
Buffer at 55ºC to 80ºC may also slightly increase the DNA.
elution efficiency. 7. Store purified DNA at -20ºC.

7. Store purified DNA at -20ºC.

EZ-10 Spin Column Handbook 16

EZ-10 Spin Column Handbook 15
 Troubleshooting Guide: EZ-10 Spin Column
PCR Products Purification Kit and DNA Gel
Extraction Kit
BINDING BUFFER II WITH PH INDICATOR
1) Low Yield
Adsorption of DNA to silica membrane depends on There are number of variables that can cause
pH, it is typically 95% if the pH is <7.0. At a higher low yield
pH binding is drastically reduced. When the a. Each step has to be strictly followed.
electrophoresis buffer has been used repeatedly, b. Make sure column binding capacity 10µg is
incorrectly prepared, or used in an enzymatic not exceeded.
reaction and is strongly basic, the agarose gel
piece containing DNA will have a high pH 2) Samples floats upon loading in agarose gel
environment. As a result, DNA will bind poorly to The sample contains ethanol from washing step.
the silica membrane. This can be illustrated in table Discard the liquid waste from the collection tube
1.1. after washing step, and spin again for additional
two minutes before the final elution step.

3) For optimal results in downstream DNA

sequencing, an additional washing step is
recommended.

PRODUCTS ARE INTENDED FOR BASIC

SCIENTIFIC RESEARCH ONLY!
NOT INTENDED FOR HUMAN OR ANIMAL USE!

EZ-10 Spin Column Handbook 17 EZ-10 Spin Column Handbook 18

Other Kits Available

EZ-200 Spin Column Plasmid DNA MediPreps Kit

BS4634 (4preps), BS464 (20preps)

EZ-500 Spin Column Plasmid DNA MaxiPreps Kit

BS4654 (4preps), BS466 (20preps)

EZ-10 Spin Column Plasmid DNA MiniPreps Kit

BS413, BS414

EZ-10 Spin Column PCR Products Purification Kit

BS363, BS364

EZ-10 Spin Column DNA Gel Extraction Kit

BS353, BS354

EZ-10 96-Well Spin Column PCR Products

Purification Kit
BS3652 (2preps), BS365 (5preps)

And much more………..

Please visit www.biobasic.com

A Leader in Serving Science

EZ-10 Spin Column Handbook 19