Sample Preparation
Guidelines for the Vacuum Prep Tool 
Introduction
In order to perform DNA analysis using Pyrosequencing™ technology, PCR products have to be
processed to yield single-stranded DNA with annealed sequencing primer. The simple and robust sample
preparation process, outlined below, produces high quality DNA from crude PCR reactions without prior
purification. The PCR product, with one strand 5’-biotinylated, is captured on streptavidin-coated beads.
After denaturation by alkali treatment, the non-biotinylated DNA strand is removed by washing. The
washing step also serves to neutralize the pH. Finally, the beads with attached, single-stranded DNA are
transferred to buffer containing the sequencing primer. After primer annealing, the DNA is ready for
analysis.
Vacuum Prep Tool preparation of template for DNA analysis using the
Pyrosequencing technology.
Additional materials needed
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Mixer/shaker for microtiter plates (room temperature)
Heating block (80C)
Vacuum source: water jet or vacuum pump (min vacuum 300 mmHg, e.g., KNF Neuberger
LABOPORT N816.12KN, order no. 055529/055305-220V/50 Hz, 055529/055307-115V/60Hz)
PSQ HS 96 Plate (40-0028) for PSQ HS 96 or PSQ 96 Plate low (40-0010) for PSQ 96MA platforms
96-well PCR plate
Strip caps or sealing tape
PSQ 96HS instruments:
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PSQ HS 96 Sample Prep Thermoplate Kit (60-0123
Additional reagents needed
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Biotinylated PCR template
Streptavidin Sepharose™ HP (5 ml, order no. 17-5113-01, Amersham Biosciences, Uppsala,
Sweden)
Binding buffer (Appendix 2)
Ethanol (70%)
Denaturation solution (Appendix 2)
Washing buffer (Appendix 2)
Annealing buffer (Appendix 2)
Sequencing primer
High purity water (MilliQ™ 18.2 MΩ x cm or equivalent)
Sample preparation for PSQ 96HS instruments:
Capture of PCR product to beads
Biotinylated PCR products are immobilized on streptavidin-coated Sepharose™ beads. Parallel
immobilization of several samples can be performed in a sealable 96-well PCR plate.
All steps are performed at room temperature unless otherwise stated.
1. Allow all solutions to reach room temperature before starting.
2. Shake the bottle with Streptavidin Sepharose™ HP beads until a homogenous solution is obtained.
3. Transfer the total required amount of Streptavidin Sepharose™ beads
(2 l per sample) to a tube.
4. Add Binding buffer to a volume equivalent to 40 l per sample. Mix.
5. In a 96-well PCR plate containing 5-10 l of a well optimized PCR reaction (at least 50% yield of a
reaction based on 0.2 M of each PCR primer), add high purity water to 40 l per well.
6. Mix with an equal volume (40 l) of the Binding buffer - Streptavidin Sepharose™ bead mix.
7. Using a mixer/shaker, incubate at room temperature for 5 min while agitating constantly to keep the
beads dispersed. It might be necessary to increase the immobilization time for longer fragments.
Tip: During the immobilization incubation, prepare the sequencing primer – Annealing buffer solutions
needed for primer annealing.
Strand separation:
Will require 5 troughs – 1) priming water, 2) 70% EtOH, 3) Denaturing (0.2M NaOH), 4) Wash buffer,
5) Wash water
8. Fill three of the troughs delivered with the Vacuum Prep Tool with approximately 180 ml of high purity
water, 70% ethanol and Washing buffer, respectively. In a fourth trough add approximately 120 ml
Denaturation solution. Refill the troughs to approximately these levels whenever needed.
9. Prime the probes of the Vacuum Prep Tool by applying vacuum and lowering the tool into the trough
with high purity water for approximately 30 seconds to wash the filter probes. If needed, the
functionality of the Vacuum Prep Tool can be tested according to Appendix 1.
10. Capture the beads containing the immobilized templates on the filter probes by slowly lowering the
Vacuum Prep Tool into the PCR plate. NOTE: Sepharose™ beads sediment quickly and the capturing
of beads must take place within three minutes after termination of the agitation!
11. Keeping the PCR plate and the Vacuum Prep Tool together, carefully lift and tilt to check that all
beads have been captured onto the probe tips. The surface of the solution should be level with or
lower than the filter probe tips, when they are resting on the bottom of the well.
12. Move the Vacuum Prep Tool to the trough with 70% ethanol and let the solution flush through the
filters for 5 seconds.
13. Move to Denaturation solution for 5 seconds.
14. Move to Washing buffer for 5 seconds.
15. Release the vacuum.
16. Release the beads in a PSQ HS 96 Plate, prefilled with 0.3 M sequencing primer in 12 l Annealing
buffer, by moving the Vacuum Prep Tool in small circles rubbing the filter probes against the bottom of
the wells.
17. Shake the Vacuum Prep Tool in a Wash Trough of high purity water to release any remaining beads.
18. If preparing more then one plate, repeat the procedure from step 9 in the Priming trough. NOTE:
This step should only be done after the shaking in Step 17. After the last plate has been
prepared, wash the filter probes with high purity water according to step 9.
19. Release the vacuum and store until use.
Primer annealing
20. Heat the plate with the samples at 80C for 2 minutes using the PSQ 96 HS Sample Prep
Thermoplate Kit. Do not forget to place the “iron” on top of the plate.
21. Remove the plate from the heating block and let the samples cool to room temperature. NOTE: If
you have
22. After reaching room temperature, continue with the sequencing reaction.
Appendix 1
Vacuum Prep Tool
Function test
Before using the Vacuum Prep Tool, a simple function test can be made.
1. Fill each of the 96 wells of a microtiter plate with 80 l high purity water.
2. Apply vacuum to the Vacuum Prep Tool by opening the vacuum switch.
3. Move the tool to the microtiter plate and lower the filter probes into the wells.
4. Wait for 10 seconds.
5. All wells should now be empty.
6. If not, exchange failing filter probes and repeat the test.
Changing filter probes
The filter probes, which can be reused at least 50 times, should be replaced when the flow rate through
the filters is reduced. The filter probes (60-0180, 100/pkg) are individually exchangeable.
Exchange by pulling out old filter probes and gently inserting new ones.
Note: Use powder-free gloves to avoid contaminating the filters.
Remove
Replace
Appendix 2
Preparation of buffers used in the Sepharose Bead Sample Preparation:
Four separate solutions are needed for the Pyrosequencing template preparation. In addition,
4 M HCl and 1 M HAc are needed for pH adjustments.
Binding buffer pH 7.6
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10 mM Tris-HCl
2 M NaCl
1 mM EDTA
0.1% Tween 20
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Chemical formula
Tris
Sodium
chloride
EDTA
Tween 20
C4H11NO3
NaCl
C10H16N2O8
polyoxyethylenesorbitanm
onolaurate
Molecular
weight
(g/mol)
121.14
58.44
Concentration in
buffer
Grams needed for
1 liter buffer
10 mM
2M
1.21
117
292.25
1 mM
0.1%
0.292
1 ml
Procedure:
1. Dissolve chemicals in 900 ml MilliQ (18.2 M x cm) water.
2. Adjust pH to 7.6 with 1 M HCl.
3. Add 1 ml Tween 20 and fill up to 1000 ml with MilliQ (18.2 M x cm) water.
Note: If a sterile filtrated buffer is desired, sterile Tween 20 should be added after filtration.
1X annealing buffer (1XAB), pH 7.6
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20 mM Tris-Acetat
2 mM MgAc2
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Chemical formula Molecular weight
(g/mol)
Concentration in
buffer
Grams needed for
1 liter buffer
Tris
Magnesium acetatetetrahydrate
C4H11NO3
C4H6MgO4x4H2O
121.14
214.46
20 mM
2 mM
2.42
0.43
Procedure:
1. Dissolve chemicals in 900 ml MilliQ (18.2 M x cm) water.
2. Adjust pH to 7.6 with 4 M Acetic acid at +22C 1C.
3. Fill up to 1000 ml with MilliQ (18.2 M x cm) water.
Denaturation Solution, 0.2 M NaOH
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Sodium hydroxide
Chemical formula Molecular weight
(g/mol)
NaOH
40.00
Final concentration Grams needed for
1 liter
0.2 M
8.00
Procedure:
1. Dissolve chemicals in 950 ml MilliQ (18.2 M x cm) water.
2. Fill up to 1000 ml with MilliQ (18.2 M x cm) water.
Washing buffer, pH 7.6
-
10 mM Tris-Acetat
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Tris
Chemical formula Molecular weight
(g/mol)
C4H11NO3
121.14
Procedure:
4. Dissolve chemicals in 900 ml MilliQ (18.2 M x cm) water.
5. Adjust pH to 7.6 with 4 M Acetic acid at +22C1C.
6. Fill up to 1000 ml with MilliQ (18.2 M x cm) water.
4M HAc for pH adjustments
Concentration in
buffer
10 mM
Grams needed for
1 liter buffer
1.21
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Chemical formula
Acetic acid
(glacial)
C2H4O2 (100%)
Molecular weight
(g/mol)
60.05
Final
concentration
4M
Milliliters needed for
200 ml solution
45.8
Procedure:
1. Add the concentrated acid to 140 ml MilliQ (18.2 M x cm) water.
2. Fill up to 200 ml with MilliQ (18.2 M x cm) water.
1M HCl for pH adjustments
Chemicals:
The chemicals should be of pro analysis quality.
Chemical
Chemical formula
Hydrochloric
acid (fuming)
HCl (37%)
Molecular weight
(g/mol)
36.46
Final
concentration
1M
Procedure:
1. Add the concentrated acid to 160 ml MilliQ (18.2 M x cm) water.
2. Fill up to 200 ml with MilliQ (18.2 M x cm) water.
Milliliters needed for
200 ml solution
16.7