Download 96-well PCR Cleanup Manual for Non

96-well
PCR Cleanup
Manual for
Non-Kit Users
2
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Microplate Components Required . . . . . . . . . . . . . . . . . . .4
Buffer Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
PCR Cleanup Procedure . . . . . . . . . . . . . . . . . . . . . . . .6
Overview of Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Vacuum Filtration Protocol . . . . . . . . . . . . . . . . . . . . . . . .7
Centrifugation Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . .9
PCR Cleanup Automation . . . . . . . . . . . . . . . . . . . . . . . .10
Product Performance . . . . . . . . . . . . . . . . . . . . . . . . . .11
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Additional Accessories . . . . . . . . . . . . . . . . . . . . . . . .14
3
Introduction
The 96-well PCR Cleanup Kit provides a rapid method for purification of
PCR products from unwanted reaction components. PCR products from
100bp to 10kb can be separated from nucleotides, salts, primers and
proteins in less than 10 minutes without the need for time-consuming
precipitations or labor intensive resin purifications. The purified DNA is
ready to use in downstream applications such as fluorescent sequencing, restriction digestions, ligations, and microarray analysis.
Microplate Components Required
Catalog No.
Description
7700-2810
DNA Binding Plate
Qty/Case
25
7701-5250
250 µL V-bottom Collection Plate
50
7701-5200
2 mL Waste Collection Plate
25
7704-0001
96-well Adhesive Plate Seals
100
Buffer Formulations
Buffer
Qty needed
(per plate)
Formulation
DNA Binding Buffer
20 mL
4M Guandine-HCI, 0.75 M Potassium Acetate, pH 4.6
Wash Buffer
10 mL
40% (v/v) 100 mM Tris, 20 mM EDTA, 0.4M NaCl,
pH 7.5. Requires addition of ethanol prior to use.
Wash Buffer to yield a final concentration of 60%
(v/v) ethanol.
Elution Buffer
10 mL
10 mM Tris, 0.1 mM EDTA, pH 7.5
All buffers listed in the table above are stable for 1 year at room temperature (15-30°C)
Equipment Required
Vacuum Filtration Protocol
• Appropriate pipettes and pipette tips
• Whatman Vacuum Manifold System (Cat. No. 7705-0107 contains
manifold with gauge, regulator, two-way control valve and Teflon®/
silicone vacuum assist) or equivalent system compatible with
Whatman plates
• 750 µL Large Spacer Plate (Cat. No. 7701-5750 or equivalent)
• Sufficient tubing and moisture trap
• Vacuum source capable of at least -15 inches Hg
Pressure Conversion Chart
Conversions from inches Hg to:
Multiply by:
Millibars (mbar)
33.86
Millimeters of mercury (torr)
25.4
Kilopascals (kPa)
3.386
Pounds per square inch (psi)
0.4912
4
Equipment Required (Continued)
Centrifugation Protocol
• Appropriate pipettes and pipette tips
• Centrifuge capable of 1800 x g
• Microplate carrier capable of accommodating 7.5 cm stack height
Precautions
• This procedure involves the handling of amplification products.
Proper care must be taken to avoid cross contamination of samples
and buffers. Change gloves often. Change pipette tips after any
contact with amplified material to avoid contamination of buffers
or samples.
• Some of the buffers listed in this manual are potential eye/skin
irritants. Proper laboratory precautions should be followed.
• This procedure is designed for research use only; not for use in
diagnostic procedures.
• MSDS sheets are available upon request.
• Whatman plates are designed to be disposable, single-use-only products.
5
96-Well PCR Cleanup Procedure
This section includes all of the information necessary to carry out the
purification of PCR products using vacuum filtration and centrifuge
systems. The protocol also can be adapted for use on liquid
handling systems.
Overview of Procedure
PCR
Product
Add Binding Buffer
Bind
Filter
Add Wash Buffer
Wash
Filter
Add Elution Buffer
Elute
Filter and Collect
6
Sample Preparation
Sample Volumes less than 25 µL
Add enough DNA Binding Buffer to bring the total volume to 75 µL
and mix by aspiration with a pipette. For example, to a 5 µL PCR
reaction, add 70 µL DNA Binding Buffer.
Sample Volumes between 25 µL and 125 µL (recommended)
Add 2 volumes DNA Binding Buffer to 1 volume of the PCR sample
and mix by aspiration with a pipette.
Sample Volumes between 125 µL and 200 µL
Add 1 volume of DNA Binding Buffer to 1 volume of the PCR
sample and mix by aspiration with a pipette.
Sample Volumes greater than 200 µL
Divide the reaction into 2 or more tubes and follow the above
criteria for sample preparation based on the new sample volume.
Note: It is not necessary to remove mineral oil from the PCR
samples. If mineral oil is being used, base the above
calculations on the aqueous sample volume only.
Vacuum Filtration Protocol
1. Prepare vacuum manifold for DNA binding.
Attach the vacuum source to the manifold. Place the 2 mL Waste
Collection Plate inside the vacuum manifold. Place the vacuum
manifold collar on top of the manifold. Place the DNA Binding
Plate on top of the vacuum collar, ensuring that the two plates
are aligned alphanumerically (A1 to A1).
2. Apply the full volume of each prepared sample directly
to the center of each well of a DNA Binding Plate.
Apply vacuum between -15 and -20 inches Hg and allow the
sample to be pulled through the filter media for 1 minute. Release
the vacuum using the manifold switch and check to see that
all liquid has been evacuated from each well. If fluid remains
in any of the wells, repeat this step until all liquid has cleared.
Note: If all 96-wells of the plate are not being processed simultaneously, the unused wells must be sealed with the vacuum
assist or adhesive sealing tape before vacuum is applied.
7
Note: Ensure that ethanol has been added to Wash Buffer.
If ethanol has not been added, refer to “Buffer Formulation”.
3. Add 400 µL Wash Buffer to each well of the DNA
Binding Plate.
Apply vacuum between -15 and -20 inches Hg, and allow the
buffer to be pulled through the filter media for 1 minute. Release
the vacuum using the manifold switch, and check to see that all
liquid has been evacuated from each well. If fluid remains
in any of the wells, repeat this step until all liquid has cleared.
4. Apply maximum vacuum for an additional 2 minutes to dry
the DNA Binding Plate.
This step removes residual Wash Buffer from the DNA Binding
Plate. Seal any unused wells of the DNA Binding plate before
drying. Ensure that the wells in use are exposed to ambient airflow
for efficient drying. Release the vacuum using the manifold switch.
Using a clean absorbent paper towel, blot the bottom of the DNA
Binding Plate to remove any residual Wash Buffer from the drip
directors. The removal of ethanol present in the Wash Buffer is
important for downstream applications.
5. Prepare vacuum
manifold for DNA
collection.
As illustrated, place
the 750 µL large
Spacer Plate inside
the vacuum manifold.
Place a 250 µL
Collection Plate on
top of the spacer plate.
Place the vacuum
collar on top of the
manifold. Place the
processed DNA
Binding Plate on top
of the vacuum collar.
Ensure that the DNA
Binding Plate and the
250 µL Collection Plate
are aligned alphanumerically (A1 to A1).
8
6. Add 100 µL Elution Buffer (or nuclease-free water)
to the center of each well of the DNA Binding Plate.
Allow the plate to stand for 1 minute. Apply vacuum between
-15 and -20 inches Hg, and allow the buffer to be pulled through
the filter media for a minimum of 1 minute. Release the vacuum
using the manifold switch and ensure that all liquid has been
evacuated from each well. If fluid remains in any of the wells
repeat this step until all liquid has cleared.
Note: If more concentrated eluate is desired, the volume of elution
buffer can be decreased to a minimum of 25 µL. However,
as elution volume is decreased, some loss in volume might
be observed due to liquid retention in the filter media.
7. Remove the 250 µL Collection Plate from the vacuum
manifold.
The purified PCR product is now suitable for use in downstream
applications. For storage, cover the 250 µL Collection Plate with
the 96-well Adhesive Plate Seal.
Centrifugation Protocol
When using the centrifugation protocol outlined below, ensure that
the centrifuge meets the criteria noted in the “Equipment Required”
section. Refer to manufacturer’s manual for operating instructions.
1. Prepare plates for DNA binding.
Place the 96-well DNA Binding Plate on top of the 2 mL Waste
Collection Plate to form a plate stack. Ensure that the two plates
are aligned alphanumerically (A1 to A1).
2. Apply the full volume of each prepared sample directly to
the center of each well of the DNA Binding Plate.
Place the stacked plates in the centrifuge tray.
3. Centrifuge for 2.5 minutes at 1800 x g.
The DNA is now bound to the filter media of the DNA binding plate.
Remove the stacked plates from the centrifuge tray.
9
Note: Ensure that ethanol has been added to the Wash Buffer. If
ethanol has not been added, refer to “Buffer Formulation”.
4. Add 400 µL Wash Buffer to each well of the DNA binding plate.
Place the stacked plates in the centrifuge tray.
5. Centrifuge for 4.5 minutes at 1800 x g.
This step removes the residual Wash Buffer from the DNA Binding
Plate. Remove the stacked plates from the centrifuge tray. Using a
clean absorbent paper towel, blot the bottom of the DNA Binding
Plate to remove any residual Wash Buffer from the drip directors.
The removal of ethanol present in the Wash Buffer is important for
downstream applications.
6. Prepare plates for DNA Collection.
Place the DNA Binding Plate on top of a 250 µL Collection Plate
to form a plate stack, ensuring that the two plates are aligned
alphanumerically (A1 to A1).
7. Add 100 µL Elution Buffer (or nuclease-free water) to the
center of each well of the DNA Binding Plate.
Allow the plates to stand for 1 minute. Place the stacked plates in
the centrifuge tray.
Note: If more concentrated eluate is desired, the volume of Elution
Buffer can be decreased accordingly. However, as elution
volume is decreased, some loss in volume might be
observed due to liquid retention in the filter media.
8. Centrifuge for 2.5 minutes at 1800 x g.
The PCR product has now been eluted into the 250 µL
Collection Plate.
9. Remove the stacked plates from the centrifuge.
The purified PCR product is now suitable for use in downstream
applications. For storage, cover the Collection Plate with the
96-well Adhesive Plate Seal.
PCR Cleanup Automation
The Whatman 96-well PCR Cleanup System is compatible with
high-throughput platforms. Automated protocols for specific high
throughput systems can be obtained through Whatman technical
service or by download at www.whatman.com.
10
Product Performance
The 96-well PCR Cleanup Kit provides an easy-to-use method for
purifying PCR products from unincorporated reaction components.
After DNA is captured onto a filter media, impurities are washed away
and the purified PCR product is eluted into an isotonic buffer. Eluted
DNA is free of polymerases and other protein adjuncts (>99.5%
removal), as well as nucleotides, salts and primers (see figure 1). The
highly purified product is ready for use in applications such as fluorescent sequencing, restriction digestion, ligation, and microarray analysis
without the need for further manipulations. High recovery of DNA
(>85% - see figure 2) has been observed for PCR products 100-10,000
bp in length with extremely reproducible results (less than 10% CV)
and no cross contamination between wells. The kits are designed for
vacuum filtration, centrifugation, and liquid handling systems, such
as the Biomek® 2000 instrument.
Figure 1. Primer Removal
Before
Cleanup
After
Cleanup
Before
Cleanup
After
Cleanup
MW
1.3Kb
Globin
213bp
Amelogenin
Primer
Bands
Product from two PCR reactions (1.3kb -globin PCR, and 213bp Amelogenin PCR) visualized on a
2% agarose gel before and after purification using the 96-well PCR Cleanup Kit. MW = Promega PCR
MW markers (1000, 750, 500, 300, 150 & 50 base pairs)
Average % Recovery
Figure 2. PCR Product Size vs. Percent Recovery
100%
90%
80%
70%
60%
50%
1800
507
213
PCR Product Size (numbers of base pairs)
Different size PCR products processed using the 96-well PCR Cleanup Kit. Each fragment size
was run n=8. Average percent recovery determined by PicoGreen® assay.
11
Troubleshooting
Problem
Low DNA Yield
Comments/Suggestions
PCR reaction not optimized.
PCR optimization studies can
increase amplification efficiency
for improved yield.
Insufficient mixing with
DNA Binding Buffer before
transferring sample to the
DNA Binding Plate.
Sample not added to the
center of the well. Sample
that is not in contact with the filter
media will not bind, resulting in
low DNA recovery.
Elution buffer not added
to the center of the well. To
ensure uniform coverage of the
filter media, Elution Buffer should
be added directly to the center of
the filter media.
Elution volume less than 50 µL.
Due to liquid retention in the filter
media, a higher percentage of
volume loss will occur with
lower elution volumes. Elution
volumes less than 50 µL are only
recommended if very high DNA
concentrations are required.
Nucleases present in the eluate.
If using an alternative elution
buffer (i.e. water), ensure that
it is nuclease-free to ensure
DNA integrity.
12
Problem
Comments/Suggestions
Wells Not Filtering
Nonuniform airflow. If processing
less than 96 wells simultaneously,
the Vacuum Assist or sealing tape
must be used on empty wells to
ensure proper vacuum.
Airlock due to bubble in the
well. Buffers should be added to
the center of each well to avoid air
bubbles. Removal of air bubbles by
sample agitation is recommended.
Insufficient vacuum. Verify that
the vacuum manifold reaches the
specified vacuum as outlined in
the protocol.
Poor DNA Performance
Ethanol in Eluate
Incomplete drying. Ensure that
the Wash Buffer drying process is
followed carefully and completely.
High Salt in Eluate
Ensure that the wash step has
been properly performed. Verify
that ethanol has been added to
Wash Buffer as outlined in
“Preparation of Buffer”.
Unexpected Size
Fragments on Gel
PCR reaction not optimized.
PCR optimization can increase
priming specificity for the
desired product.
Sample cross contamination
or buffer contamination. Proper
molecular biology precautions
must be maintained when handling
amplified products. All pipette tips
must be changed after contacting
amplification products.
13
Additional Accessories
Catalog No.
Description
Qty/Case
7705-0107
Vacuum Manifold System
(contains: Manifold with gauge,
regulator, two-way control valve,
and Teflon®/silicone vacuum assist)
1 per/box
Teflon/Silicone Vacuum Assist
(replacement for vacuum assist
contained in Vacuum Manifold System)
6 per/pack
7701-5200
2 mL Waste Collection Plate
25 per/case
7725-0118
96-well Filtrate Directors
(for Biomek® 2000 processing)
25 per/case
7705-0120
Small Whatman Vacuum
Adapter Collar for Biomek 2000
1 per/pack
7705-0121
Medium Whatman Vacuum
Adapter Collar for Biomek 2000
1 per/pack
7705-0205
Notice
The information in this document is given in good faith, and is for guidance only. It is not intended to
override or void any of Whatman’s Standard Conditions of Sale. Those Conditions remain in full and
complete effect, and govern all relations between Whatman and the purchaser of the goods which
relate to this document.
PCR is a patented process covered by U. S. Patents 4,683,195 and 4,683,202 and foreign equivalents
owned by Hoffman-La Roche AG. PicoGreen is a registered trademark of Molecular Probes, Inc.
Biomek® is a registered trademark of Beckman Coulter, Inc.
Teflon® is a registered trademark of DuPont
Whatman® is a registered trademark of Whatman Inc.
©Copyright, Whatman Inc., 2002 Printed in USA
14
15
Whatman
www.whatman.com
For pricing and availability, please contact:
USA
Japan
Whatman Inc.
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Clifton, NJ 07014
Toll Free: 800-WHATMAN
Fax: 973-773-0168
E-mail: [email protected]
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Tokyo 110-0005 Japan
Tel: +81 3 3832 6707
Fax: +81 3 3832 6457
E-mail: [email protected]
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St. Leonard’s Road
20/20 Maidstone
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ME 16 OLS
Tel: +44 (0) 1622 676670
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E-mail: [email protected]
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Tel: + 65 6534 0138
Fax: + 65 6534 2166
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India
China
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Shanghai 200032
P.R.China
Tel: 86 21 6443 7176
Fax: 86 21 6443 2568
E-mail: [email protected]
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V.N.Purav Marg, Chembur
Mumbai 400071
India
Tel: 91 22 529 7035
Fax: 91 22 529 7036
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The Advantages Filter ThroughSM
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www.whatman.com
Part No. 69I0030019, Rev. A