384-Well PCR Purification Using the DNA Binding - Whatman

Whatman® Application Note
Introduction
The purification of PCR products is a commonly used
practice in many of today’s molecular biology labs. The
purpose of the cleanup step is to purify PCR amplification
products away from undesirable reaction components
such as salts, excess nucleotides, primers, adjuncts
and proteins (e.g., Taq polymerase). The removal of
these components improves the reliability of downstream
analytical methods routinely performed in genomics
labs, thereby increasing process flow and decreasing
handling costs.
The Whatman 384-Well DNA Binding Plate contains a
unique membrane engineered for the high throughput
preparation of double-stranded DNA. This plate, together
with the simple method described below, will provide a
high throughput, low cost procedure for purifying PCR
products for downstream applications such as DNA
sequencing or microarray printing.
Materials
The Whatman plates used in this application are
O 384-Well DNA Binding Plate (Cat. No. 7700-2110)
O 384-Well 400µL Waste Collection Plate
(Cat. No. 7701-5400)
O 384-Well 100µL Collection Plate
(Cat. No. 7701-1100)
The solutions used in this application are
O DNA Binding Buffer 20% 8M Guanidine
Hydrochloride: 80% Ethanol (v/v)
O Wash Buffer 20% 100mM Tris, 20mM EDTA, 0.4M
NaCl, pH 7.5: 80% Ethanol (v/v)
O Elution Buffer 10mM Tris, 0.1mM EDTA, pH 7.5
Alternatively, nuclease free water can be used for elution
of the PCR amplification products. Other equipment
required for this protocol include a microplate centrifuge
capable of accommodating a 5.85cm (2.3 inch) stack
height, a pipetting system and commercially available
aerosol resistant pipet tips (>100µL fill volume).
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Techniques and Technical Insight to Optimize Use of Whatman Products
384-Well PCR Purification Using
the DNA Binding Filter Plate
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Methods
PCR Amplification
Amelogenin PCR product (213bp) was amplified using the
Promega GenePrint ® Sex ID kit. Two fragments from
pGEM/luc plasmid (500bp and 1.9kb) were also amplified
using custom primers and Promega PCR reagents.
PCR Cleanup Procedure
This procedure can be performed on any liquid handling
robot and uses a 384-well purification format adaptable for
processing 5 to 30µL PCR reactions from either 96 or
384-Well thermal cycling plates. The method described here
was carried out on a Biomek ® 2000 (Beckman Coulter)
liquid handling robot and the GP8 centrifuge from Thermo IEC.
96-Well PCR Plates containing 25µL amplified DNA were
placed on the Biomek 2000 work surface equipped with left
and right side modules, buffer reservoirs, and the MP200
pipetting tool (8-channel). DNA Binding Buffer (60µL) was
added directly to each amplification reaction and mixed 4
times using a medium pipetting speed and a 60µL mix
volume. The contents were then transferred to the 384-Well
DNA Binding Plate using a slow aspirate and dispense
speed followed by a 10µL blowout in order to maximize
volume transfer.
For reactions performed in 384-Well PCR Plates or plates
with working volumes less than 85µL, 35µL of the DNA
Binding Buffer was first added to each well of the 384-Well
DNA Binding Plate. The PCR mixture was then transferred
from the PCR plate to the wells of the DNA Binding Plate.
The wells of the PCR plate were rinsed with 25µL of DNA
Binding Buffer, which was added to the corresponding wells
of the DNA Binding Plate. The contents of the wells were
mixed 3 times using a medium pipetting speed and a mix
volume equal to 40% of the total solution volume. A postmixing
delay of 2 - 5 seconds with a 10µL blow out was
performed to completely clear the viscous liquid from the
pipet tips.
The 384-Well DNA Binding Plate stacked on top of a 400µL
Waste Collection Plate was placed in the centrifuge and
filtered at 1,800xg for 2.5 minutes. Two sequential wash
steps were then performed, each through automated
addition of 90µL of the Wash Buffer followed by centrifugation
at 1,800xg at 2.5 and 5 minutes respectively. The
purified DNA was eluted by adding 50µL of Elution Buffer to
each well, incubating for 1 minute and centrifuge filtering
(1800xg for 2.5 minutes) into a 100µL collection plate.

DNA Quantitation for Recovery Evaluation
The PicoGreen ® detection systems (Molecular Probes) was
used to quantitate double-stranded PCR product. Percent
recovery was calculated by comparison of purified vs.
unpurified PCR product quantitation.
DNA Sequencing
DNA sequencing was performed using BigDye Terminator
reaction chemistry (v 2.0) and the ABI Prism ® 3100 Genetic
Analyzer. Amplification was conducted at 1/4 strength by
combining 2µL purified amplicon and 8µL deionized H 2O
(containing 1.6 pmol primer) and 2µL BigDye reagent.
Purity of PCR Products after Cleanup
Primer removal: The OliGreen ® single-stranded DNA detection
system from Molecular Probes was used to quantitate
initial and final primer concentrations for PCR reactions.
Protein removal: PCR reactions containing varying
concentrations of BSA or Taq polymerase were tested
before and after PCR cleanup using the MicroBCA reagent system from Pierce Immunochemicals.
Nucleotide removal: Nucleotide concentrations were
measured spectrophotometrically at 260nm using reactions
devoid of primers, target DNA and Taq polymerase.
Results
DNA Recovery and Consistency
Amplified DNA fragments of varying size were purified
using the Whatman 384-Well DNA Binding Plate and the
protocol outlined in the “Methods” section. Recovery of
the fragments exceeded 90% in all instances (Figure 1).
Recovery for PCR mixtures, distributed evenly across the
384-Well DNA Binding Plates, exhibited low variability from
well to well (< 8% CV) ensuring a standard purification
performance across the entire plate. Using the Biomek 2000
equipped with an 8-channel pipetting head the processing
times were approximately 1:25:10 and 2:36:50 for 2 and
4 plates, respectively.
5.00 100.0%
4.50
4.00
3.50
Crude Input (µg)
DNA Yield (µg)
% Recovery
98.0%
96.0%
3.00
2.50
94.0%
2.00
92.0%
1.50
90.0%
1.00
0.50
88.0%
0.00
213bp Amelogenin 500bp pGEM
PCR Product
1.9kb pGEM
86.0%
Figure 1. PCR product pools for 3 different fragments run in parallel
using the Whatman 384-Well PCR Cleanup Method on the Biomek 2000.
Recoveries were determined by Picogreen analysis of input vs purified
PCR products.
DNA Quantity (µg/well)
% Recovery
Well to Well Discrimination
Critical to multiwell applications is the proof that well to
well crosstalk does not occur. To illustrate this point, a
1.9kb pGEM insert was amplified and subsequently
purified in alternate wells in a 384-Well DNA Binding Plate.
Buffer was added to the intervening wells as a blank.
Following the PCR cleanup procedure, all wells were
reamplified using sensitive nested PCR primers. If crosstalk
had occurred then the wells receiving buffer would
amplify a product. As can be seen in Figure 2, only the
wells that contained the true PCR product were amplified.
Figure 2. Alternating positive and negative PCR purification
(1.9kb pGEM insert PCR product). An aliquot of each eluate was
reamplified in a 25µL PCR reaction with nested primers. The
nested PCR was run on a 1% agarose gel and visualized with
ethidium bromide.
Effects of Adjuncts on PCR Cleanup
To enhance PCR processivity or to alleviate problems
due to template sequence, adjuncts are added to the
PCR mixture. Common additives such as DMSO,
glycerol and betaine, while enhancing the amplification
reaction, can interfere with downstream PCR cleanup.
Betaine is known to interfere with silica-based cleanup
procedures resulting in low yield and recovery.
To test the effects of these additives on double-stranded
DNA binding to the 384-well DNA Binding Plate, we
added increasing quantities of herring sperm DNA in
standard PCR buffer conditions (10 mM Tris, pH 9,
50 mM KCl,1.5 mM MgCl 2 and 0.1 % Triton X-100)
to the DNA Binding Plate in the presence or absence
of 2% DMSO, 1.5M Betaine or 0.1% Glycerol. As can
be seen in Figure 3, DMSO and Glycerol have no effect
on binding of DNA to the DNA Binding Plate. Betaine
caused a slight reduction (~15%) in yield.
An important observation from this study is the high
binding capacity of the 384-Well DNA Binding Plate
which is > 7µg/well.

DNA Yield (µg/well)
Effect of Additives on 384-Well PCR Cleanup
10
9
8
7
6
5
4
3
2
1
0
0 2 4 6 8 10
HS DNA Input (µg/well)
Standard PCR 2% DMSO 1.5M Betaine 0.1% Glycerol
Figure 3. Herring Sperm DNA was added to the wells of the 384-Well
DNA Binding Plate in the presence an absence of additives. DNA
recovery was measured following elution using PicoGreen.
Removal of PCR Contaminants
Important for downstream processing is the removal of
excess primers, nucleotides and protein contaminants
from the PCR product. Contaminants are hazardous for
automated sequencing and for microarray analysis of
PCR amplicons.
The Whatman 384-Well PCR Cleanup protocol was
designed to eliminate contaminants in the final PCR
product to ensure their optimal use in downstream
processing. The Whatman protocol removes > 92%
and 100% of the excess primers and nucleotides,
respectively (Figure 4). Critical for microarray spotting
of PCR products, the 384-Well PCR Cleanup protocol
removes > 95% of proteins including the 94 Kd Taq DNA
polymerase routinely retained by ultra-filtration methods.
% Removal
102%
100%
98%
96%
94%
92%
90%
88%
86%
Removal of PCR Contaminants
Primer removal Protein removal Nucleotide removal
Figure 4. Removal of unincorporated PCR components
DNA Sequencing
To further demonstrate the purity of the PCR products
isolated with the Whatman protocol and 384-well DNA
Binding Plate, we sequenced fragments via sensitive
capillary sequencing. It is widely accepted that contaminating
salts and residual alcohol will interfere with DNA sequencing
reactions. Good quality sequencing results were obtained
using the 1.9 kb PCR product as a template (Figure 5). We
routinely achieved read lengths in excess of 750 bp with 99%
base calling accuracy indicating high template purity.
Figure 5. ABI 3100 pherograph of a 1.9kb PCR fragment purified
with the 384-Well DNA Binding Plate and labeled with Big Dye
reaction mix.
Discussion
We have shown the validity and the utility of the Whatman
384-Well DNA Binding Plate for purification of PCR
products. The method is based on the commonly used
silica “bind, wash and elute” process that has been widely
accepted as yielding highly purified DNA for analysis.
Adaptable to many commonly used liquid handling robots,
this method is automation friendly. Using an indexing
centrifuge as part of the robotics platform, the method is
truly a “walk away” system.
The 384-Well DNA Binding Plate provides a robust support
for isolating DNA fragments for downstream processing.
The method is reliable and consistent across the entire
plate and does not appear to be affected by agents known
to interfere with 96-well silica binding, such as betaine.
High yields of DNA are routinely recovered free of excess
reaction components. The method is quick and requires
easy to prepare buffers made with chemicals routinely
found in most molecular biology labs. Bulk packaging of
plates allows a flexible and cost effective means of PCR
cleanup.
More information on automated protocols can be found at
www.whatman.com/technical support/applications.

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PCR is covered by US patents issued to Hoffman-LaRoche, Inc. All other trademarks acknowledged.
FOR RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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