Laboratory protocol for manual purification of DNA
from whole sample
For the purification of genomic DNA from the Oragene® and ORAcollect® families of collection kits.
Visit our website at www.dnagenotek.com for any additional languages and protocols.
Note: This protocol requires the use of a centrifuge capable of generating at least 3,500 × g to obtain
optimal results.
The procedure is described for purifying DNA from the entire collected sample (approximately 1 mL
to 4 mL total volume). The volumes shown should be adjusted for the actual collected volume.

Reagents included•

prepIT®•L2P (catalog #: PT-L2P)

Equipment and reagents
•
•
•
•
•
•
•
•

Centrifuge that accommodates 15 mL tubes, and is capable of generating at least 3,500 × g (see Table 2)
15 mL conical polypropylene tubes (e.g., BD Falcon #352196)
Microcentrifuge capable of running at 15,000 × g (optional)
1.5 mL microtubes (e.g., Axygen #MCT-150-C)
Air or water incubator at 50°C
Ethanol (95% to 100%) at room temperature
Ethanol (70%) at room temperature
DNA storage buffer: TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or similar solution

Pre-purification check
Weigh the sample to estimate the amount of saliva provided by the donor (see Table 1; not required for OC-100
and OCR-100). The amount of saliva collected is directly proportional to the amount of DNA recovered. As an
example, if a donor has provided less than 2 mL of saliva, you should expect to recover a lower total yield fromthis sample. Correspondingly, a donor providing more than 2 mL of saliva should result in higher total yield.
Weight of kit (without sample)
Once a sample arrives at the lab, we suggest
weighing the sample to estimate if the right amountof saliva was provided by the donor. You can expect
some variability across donors as determined byweight of triggered kit with sample. The average
weight of an empty kit is provided (Table 1). To
calculate the amount of sample collected (assuming
1 g/mL), perform the following subtraction:
Weight of kit containing sample - Weight of kit
without sample = Amount of sample collected

PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Table 1

Product #Weight of kit
without sample

OG-250/OGR-250

14.15 gOG-500/ OGD-500/OGR-500

6.81 gOG-510/ OGD-510

5.83 gOG-5205.41 gOG-575/ OGD-575/OGR-575

5.66 gON-5006.47 gwww.dnagenotek.com • support@dnagenotek.com

Procedure
Purification steps

Notes

1. Mix the sample in the DNA Genotek kit by
inversion and gentle shaking for a few seconds.

• This is to ensure that viscous samples areproperly mixed.

2. Incubate the sample at 50°C in a water incubator
for a minimum of 1 hour or in an air incubator fora minimum of 2 hours.

• This heat-treatment step is essential to maximize
DNA yield and ensure that nucleases arepermanently inactivated.
• This must be done in the original collection tube.
• The sample may be incubated at 50°C overnight
if more convenient.
• This incubation step may be performed at any timeafter sample is collected and before DNA is purified.
• A longer time is required in an air incubator
because temperature equilibration is slower thanin a water incubator.

Note: The use of an air incubator may be preferable
since the sample tubes may float in a water bath.
If a water bath must be used, ensure the samplecontaining portion of the tube remains immersed
in water.

3. Transfer the entire sample to a 15 mL centrifuge
tube (Figure 1). Note the volume of the sample.

• Transfer can be carried out either by pouring
or by pipetting with a glass or plastic pipette.

Figure 1: Before proceeding to Step 4, ensure that the entire
sample has been incubated and transferred to a fresh 15 mL
centrifuge tube, as shown.

4. Add 1/25th volume of PT-L2P and mix by
vortexing for a few seconds. (Figure 2)• e.g., 160 µL for 4 mL of sample to each tube.
• The sample will become turbid as impurities
and inhibitors are precipitated.

Figure 2: After adding the PT-L2P and incubating on
ice for 10 minutes, the sample will no longer look clear,but rather a cloudy solution.

5. Incubate on ice for 10 minutes.

PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

• Room temperature incubation can be substituted
but will be less effective at removing impurities.

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Purification steps

Notes

6. Centrifuge at room temperature for 10 minutes at
as high a speed as is possible. Minimum 3,500 x g.
Any centrifuge, either swing-out bucket or angle
rotor that can generate this g-force is suitable.

• Higher centrifugal force minimizes the amountof turbid material that will be carried over into
the purified DNA (Figure 3).
• Before proceeding, you should verify withthe tube manufacturer that the 15 mL centrifuge
tubes can withstand the centrifugal force.
• A longer period of centrifugation (up to
20 minutes) can be carried out where reducing
the turbidity of the final DNA solution isconsidered to be important.

SupernatantPellet of turbid
impurities
Figure 3: After centrifugation, there will be an accumulationof turbid material at the base of the tube. The supernatantshould be visibly clear.

7. Carefully transfer the majority of the clear
supernatant with a pipette to a fresh 15 mL
centrifuge tube. Discard the pellet.

• Leave a small volume of the supernatant behind
to avoid disturbing the pellet. The pellet contains
turbid impurities.

8. Add 1.2x volume of room temperature 95% to 100%
ethanol to the clear supernatant. Mix gently byinversion 10 times.

• During mixing with ethanol, the DNA will be
precipitated. Precipitated DNA may appear as aclot of DNA fibres (Figure 4). Even if no clot is seen,
DNA will be recovered in the following steps.

Figure 4: After the addition of ethanol, the DNA will precipitate
which may result in a visible clot of fibres.

9. Let the sample stand at room temperature for10 minutes to allow the DNA to fully precipitate.

• Incubation at -20°C is not recommended because
impurities may co-precipitate with the DNA.

10. Centrifuge at room temperature for 10 minutes at
as high a speed as is possible. Minimum 3,500 × g.

• A minimum centrifuge speed of 3,500 × g(see Table 2) is required. Any centrifuge, either
swinging-bucket or angle rotor that can generate
this g-force is suitable.

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3

Purification steps

Notes

11. Carefully remove the supernatant with a glass orplastic pipette and discard it. Take care to avoid
disturbing the DNA pellet.

• The supernatant may contain impurities andshould be removed as completely as possible.
• Precipitated DNA will be found as a pellet at thebottom of the tube and possibly as a smear down
the side of the tube (Figures 5).
• The DNA smear may be located on the side of the
tube facing away from the centre of the centrifuge.
• A smear can be located using the “scratch” test.
You can check for the presence of a DNA smear byscratching the inside of the tube using a P1000 tip.
A smear, as shown in figure 5, may be visible.

DNA may besmeared along theside of the tube.

Carefully remove
the supernatantby pipetting along
the side opposite
to the DNA smear.

DNA may also
accumulate as apellet at the baseof the tube.

Figure 5: Using a pipette tip to gently scratch along theinside of the tube may reveal the presence of a DNA smear.

12. Ethanol wash:
Carefully add 1 mL of 70% ethanol to the tube
without disturbing the smear or the pellet. Let it
stand at room temperature for 1 minute. Gently
swirl and completely remove the ethanol, being
careful not to disturb the pellet and the smear.

• It is important to remove all ethanol from thesample. Carryover of ethanol may impact
the performance of the assay.
• Take care not to disturb the DNA pellet or
the smear.
• A short centrifugation (less than 1 minute) can be
performed to facilitate complete removal of thesupernatant.
• Should the pellet detach after the ethanol wash
step, centrifuge the sample for 5 minutes at as
high a speed as is possible. Minimum 3,500 x g.

13. Rehydrate the DNA by adding 0.2 – 1 mLof TE solution and by vortexing the sample
for 30 seconds.

• If a higher concentration of DNA is desired, thevolume of TE may be reduced. A minimum of
200 µL TE solution should be used.
• Excessive drying of the pellet (> 10 minutes) andusing less than 500 µL of TE solution can make it
difficult to rehydrate (dissolve) the DNA and may
decrease the yield or make quantification difficult.
• Precipitated DNA will be found as a pellet at thebottom of the tube and possibly as a smear down
the side of the tube.
• To ensure maximum DNA recovery, the sample
must be vortexed after the addition of DNA solvent(TE solution). Vortexing will ensure that the DNA
smeared on the side of the tube is recovered(Figure 6).
• Do not hesitate to vortex the sample as the DNA
will remain high molecular weight.

For OC-100 and OCR-100, rehydrate the DNA
by adding 0.2 mL of TE solution and vortex thesample for 30 seconds.

Figure 6: Vortexing the sample for 30 seconds will allow you
to recover DNA smeared on the side of the tube. The DNA willremain high molecular weight.

PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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4

Purification steps

Notes

14. To ensure complete rehydration of the DNA(pellet and smear) incubate at room temperature
overnight followed by vortexing or at 50°C for
1 hour with occasional vortexing.

• Incomplete rehydration of the DNA is a cause of
inaccuracy in estimating DNA concentration andpotential failure of downstream applications suchas PCR.

15. Transfer the rehydrated DNA to a 1.5 mL
microcentrifuge tube for storage.
Optional step:a) Centrifuge the rehydrated DNA at room
temperature for 15 minutes at 15,000 x g.
b) Transfer the supernatant to a fresh 1.5 mL
microcentrifuge tube without disturbing
the pellet.

Note that the pellet contains insoluble, turbid material.

16. Options for storage of the fully rehydrated DNA:a) Recommended in TE, in aliquots at -20°C
for long-term storage, orb) In TE at 4°C for up to 2 months.

• Freezing of purified DNA in TE may cause
the DNA to precipitate. When thawing frozen
purified DNA, pay careful attention to rehydration,
as discussed in step 14.

• To maximize DNA recovery, ensure that the
DNA is completely rehydrated (step 14) prior
to performing this centrifugation step.
• This centrifugation step ensures that any
remaining turbid material is removed from the
DNA sample.
• Care should be taken not to disturb the pellet
when transferring the clear supernatant to a
fresh tube.

Quantification of DNA
By fluorescence method
Assays that use fluorescent dyes are more specific than absorbance at 260 nm for quantifying the amountof double-stranded DNA (dsDNA) in a DNA sample. We recommend using fluorescent dyes such as PicoGreen®
or SYBR® Green I to quantify dsDNA since there is less interference by contaminating RNA. An inexpensive
protocol using SYBR Green I is described in PD-PR-075, DNA quantification using SYBR Green I Dye and a
micro-plate reader1. Alternatively, commercially available kits such as Invitrogen’s Quant‑iT™ PicoGreen dsDNA
Assay Kit (Cat. No. Q-33130) can be used. For either protocol, we recommend that the purified DNA be diluted
1:50 with TE solution and that 5 µL be used in the quantification assay.

PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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5

By absorbance method
If you choose to quantify DNA by absorbance, we recommend that you first treat the purified sample with
RNase to digest contaminating RNA and then remove the RNA fragments by ethanol precipitation of the DNA.
A detailed protocol is described in PD-PR-040, RNA removal by double-RNase digestion2. Please note that DNA
from oral sample typically contains appreciably more RNA than found in blood samples. Ensure that alcoholprecipitated DNA is fully dissolved before reading the absorbance.
Conversion factor: An absorbance of 1.0 at 260 nm corresponds to a concentration of 50 ng/µL (50 µg/mL) for
pure dsDNA.
Ensure that absorbance values are within the linear range of the spectrophotometer. Re-dilute and re-measuresamples that fall outside of the linear range. See your instrument documentation for more information.
Method:1. Dilute a 10 µL aliquot of purified RNase treated DNA with 90 µL of TE (1/10 dilution). Mix by gently
pipetting up and down. Wait for bubbles to clear.
2.

Use TE in the reference (blank) cell.

3.

Measure absorbance at 320 nm, 280 nm and 260 nm.

4.

Calculate corrected A280 and A260 values by subtracting the absorbance at 320 nm (A320) from theA280 and A260 values.

5.

DNA concentration in ng/µL = corrected A260 × 10 (dilution factor) × 50 (conversion factor).

6.

A260/A280 ratio: Divide corrected A260 by corrected A280.

Example1.

Assume the measured A320= 0.025, A280= 0.175 and A260= 0.295

2.

The DNA concentration of the undiluted sample will be:(A260 - A320) × 10 [dilution factor] × 50 [conversion factor]= (0.295 - 0.025) × 10 × 50
= 0.270 × 10 × 50
= 135 ng/μL or 135 μg/mL

3.

The corrected A260/A280 ratio will be:(A260 - A320) ÷ (A280 - A320)= (0.296 - 0.025) ÷ (0.175 - 0.025)= 0.270 ÷ 0.150= 1.80PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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Table 2: Calculation of g-force from radius and speed of rotor
In order to generate a minimum g-force of 3,500 x g, you must select a spin speed (RPM) that is appropriate
for the size of your rotor. Highlighted below are the combinations of spin speed and rotor radius that will
generate the minimum g-force required.
e.g., If your centrifuge has a rotor radius of 10 cm you must select a minimum spin speed of 6,000 RPM.
Remember that 3,500 x g is the minimum requirement. You should spin your samples at as high a speed
as your centrifuge can support, and producing a force that your tubes can withstand.
Radius of rotor (cm)
RPM

7

8

9

10

11

12

13

14

15

16

17

18

4,000

1,253

1,432

1,611

1,790

1,049

1,969

2,148

2,328

2,507

2,865

3,044

3,223

4,500

1,586

1,813

2,039

2,266

2,493

2,719

2,946

3,172

3,399

3,626

3,852

4,079

5,000

1,958

2,238

2,518

2,798

3,077

3,357

3,637

3,917

4,196

4,476

4,756

5,036

5,500

2,369

2,708

3,046

3,385

3,723

4,062

4,400

4,739

5,077

5,416

5,754

6,036

6,000

2,820

3,223

3,626

4,028

4,431

4,834

5,237

5,640

6,043

6,445

6,848

7,251

6,500

3,309

3,782

4,255

4,728

5,201

5,673

6,146

6,619

7,092

7,564

8,037

8,510

7,000

3,838

4,386

4,935

5,483

6,031

6,580

7,128

7,676

8,225

8,773

9,321

9,870

7,500

4,406

5,036

5,665

6,294

6,924

7,553

8,183

8,812

9,442

10,071

10,70011,330

8,000

5,013

5,729

6,445

7,162

7,878

8,594

9,310

10,026

10,742

11,45912,175

12,891

8,500

5,659

6,468

7,276

8,085

8,893

9,702

10,51011,31912,127

12,936

13,744

14,553

9,000

6,345

7,251

8,158

9,064

9,970

10,877

11,783

12,68913,596

14,502

15,40916,315g-force

References
1
2

DNA quantification using the Fluorescence/DNase (F/D) assay. Replaced by DNA quantification using SYBR Green I dye
and a micro‑plate reader. DNA Genotek. PD-PR-075.
RNA removal by double-RNase digestion. DNA Genotek. PD-PR-040.

Technical support is available Monday to Friday (9h00 to 17h00 EST):•••

Toll-free (North America): 1.866.813.6354, option 6
All other countries: 613.723.5757, option 6
Email: support@dnagenotek.com

Oragene®•DNA and ORAcollect®•DNA are not available for sale in the United States.
Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
Some DNA Genotek products may not be available in all geographic regions.
®Oragene, prepIT and ORAcollect are registered trademarks of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

PD-PR-015 Issue 9/2012-02© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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7Quick reference guide:
Laboratory protocol for manual purification of DNA from whole sample
1 mL

1.5 mL

2.0 mL

2.5 mL3 mL

3.5 mL

4 mL

prepIT•L2P

40 μL

60 μL

80 μL

100 μL120 μL140 μL160 μL

95 % to 100% EtoH

1.2 mL

1.8 mL

2.4 mL3 mL

3.6 mL

4.2 mL

4.8 mL

70 % EtoH

1 mL

TE

0.2 mL to 1 mL
Purification steps

1. Mix the sample in the DNA Genotek kit by inversion and gentle shaking for a few seconds.
2. Incubate the sample at 50°C in a water incubator for a minimum of 1 hour or in an air incubator
for a minimum of 2 hours.
3. Transfer the entire sample to a 15 mL centrifuge tube. Note the volume of the sample.
4. Add 1/25th volume of PT-L2P and mix by vortexing for a few seconds.
5. Incubate on ice for 10 minutes.
6. Centrifuge at room temperature (RT) for 10 minutes at as high a speed as is possible. Minimum 3,500 x g.
7. Carefully transfer the majority of the clear supernatant with a pipette to a fresh 15 mL centrifuge tube.
Discard the pellet.
8. Add 1.2x volume of RT 95% to 100% ethanol to the clear supernatant. Mix gently by inversion 10 times.
9. Let the sample stand at RT for 10 minutes to allow the DNA to fully precipitate.
10. Centrifuge at RT for 10 minutes at as high a speed as is possible. Minimum 3,500 × g.
11. Carefully pipette off the supernatant and discard it. Take care to avoid disturbing the DNA pellet.
12. Add 1 mL of 70% ethanol to the tube without disturbing the pellet or smear. Let it stand at RT for
1 minute. Gently swirl and completely remove the ethanol, without disturbing the pellet or smear.
13. Rehydrate the DNA by adding 0.2 – 1 mL of TE solution and vortexing the sample for 30 seconds.
For OC-100 and OCR-100, rehydrate the DNA with 0.2 mL of TE.
14. Incubate overnight at RT or at 50°C for 1 hour.
15. Vortex sample and transfer the rehydrated DNA to a fresh 1.5 mL microcentrifuge tube for storage.
16. Optional: Centrifuge DNA at RT for 15 minutes at 15,000 x g then transfer the supernatant to a fresh
1.5 mL microcentrifuge tube without disturbing the pellet.
17. Storage: In aliquots at -20°C for long-term storage (recommended) or at 4°C for up to 2 months.

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8