From turbidity to clarity: Simple methods to improve theA260/A280 ratio of Oragene®-purified DNA samples
R.M. Iwasiow and H.C. Birnboim
DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

In general, DNA extracted from Oragene®/salivasamples† has a A260/A280 ratio of >1.60. Occasionally,
the ratio is lower than this, presumably due to failure
to remove food particles from the mouth before
delivering saliva. DNA samples with a lower A260/
A280 ratio regularly perform well in downstream
applications such as conventional PCR. Despite thefact that sample turbidity (i.e., absorbance at A320)
does not affect down-stream applications, some users
have enquired about modifications to our standard
protocol that will improve this ratio. In thisdocument, we describe 3 alternative methods thatcan be used to obtain high A260/A280 ratios from allsamples.

Introduction
The Oragene self-collection kit is a non-invasive
method for collecting large amounts of high quality
DNA. Since Oragene/saliva samples are stable atambient temperature for long time periods, there isno urgency to send samples to the laboratory to purify
the DNA. Once in the laboratory, DNA can be easilyand quickly extracted without a requirement for
specialized kits or equipment. The Oragene/saliva
samples need only to be heated, treated with theprepIT™•L2P and ethanol precipitated. The resulting
DNA has an A260/A280 ratio >1.6. Occasionally,
Oragene/saliva samples may contain foodstuffs fromindividuals who did not abstain from eating or
drinking 30 minutes prior to delivering the saliva.
Some foodstuffs may contribute particulates resulting
in turbid samples, which can then lead to anincreased apparent absorbance at 260 nm and
280 nm, and a lower A260/A280 ratio. In this report,
we describe how to correct the A260/A280 ratio forturbid samples. Furthermore, we describe additional
steps that can be used to eliminate turbid material
and thereby improve the A260/A280 ratio.
†Materials and methods
Saliva collection
Two donors provided 2 mL of saliva using theOragene self-collection kit. Sample 1 was collectedfrom an individual immediately after eating lunch
while Sample 2 was collected from an individual
immediately after drinking coffee. Both individuals
were specifically asked not to rinse their mouth prior
to delivering saliva. The Oragene kit was then capped
and the entire sample was mixed by inversion,
allowing the DNA to be released and stabilized.

Sample treatment
As described in the standard prepIT•L2P protocol1,samples were incubated in a water bath for 1 hour
at 50°C. A 500 µL aliquot was immediately removed
from each sample.

DNA purification
The standard prepIT•L2P purification protocol wasfollowed. In brief, 20 µL (1/25th volume) of prepIT•L2P
was added to each 500 µL sample. Each tube wasmixed well and incubated on ice for 10 minutes.
The tubes were centrifuged at 15,000 × g (13,000 rpm)
for 5 minutes. The supernatant was removed andtransferred to a fresh tube; an equal volume of 95%
ethanol at room temperature was added. The sample
was mixed by inversion and allowed to stand for
10 minutes at room temperature. The sample wascentrifuged at 15,000 × g (13,000 rpm) for 2 minutes
and the supernatant was removed and discarded.
The DNA pellet was dissolved in 100 µL of TE buffer.

Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

Some DNA Genotek products may not be available in all geographic regions, contact your sales representative for details.

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DNA analysis
DNA from the purified samples was quantified using
a fluorescent dye, SYBR® Green I. In brief, DNAsamples were diluted and 5 µL was transferred to a
PCR tube containing 1.25 µL of 20× buffer (0.1 MMgCl2, 0.02 M CaCl2 and 0.4 M Tris, pH 8.0), 5 µL of
5 mg/mL BSA, 0.125 µL SYBR Green (Sigma:S-9430,100×), and 13.63 µL of water. The unknown samples
were run with a standard curve using known amountsof reference DNA, ranging from 0 ng to 12.5 ng perassay. The samples were analyzed on a Corbett
Research Rotor-Gene RG‑3000A. The absorbance
spectrum from 220 nm to 320 nm was measured
using a Cary 1E Spectrophotometer. The A260/A280
ratio was corrected by subtracting the A320 value
from the A260 and A280 values. The molecular weightof the DNA and PCR products was determined byelectrophoresis on a 0.8% agarose gel.

of the low amount of protein or other soluble
material in the purified sample that could absorb at
280 nm. Some spectrophotometers allow the user toautomatically subtract the A320 value from their A260
and A280 readings, while others require the user to takethe A320 reading and manually subtract it from the A260
and A280 values. Regardless of the spectrophotometerused or the turbidity of sample, subtracting theA320 value is good practice and will result in a more
precise assessment of the purified sample.

Results and discussion
Improving the A260/A280 ratio by subtractionof the A320 value:
Purified DNA samples that contain turbid material
can be assessed by determining their apparent
absorbance at 320 nm; this value is due to lightscattering and not due to true absorbance. As seenin Figure 1, the spectrum of Sample 1 demonstrates
a turbid sample with an A320 value of 0.234.
When this value is subtracted from all values acrossthe spectrum according to g(χ) = f(χ) – A320, theresulting normalized spectrum in Figure 1 provides
us with “corrected” A260 and A280 values. Corrected
A260 and A280 values eliminate the contribution of
turbid material to the absorbance spectrum and allow
a more precise assessment of the true A260/A280 ratio.
Since the same A320 value is subtracted across theentire spectrum, the percent contribution varies at
each point along the curve. For example, in Sample 1
at A280 (0.576), the A320 (0.234) contributed 40.6% tothe value. However, at A260 (0.877), the A320 (0.234)
only contributed 26.7% to the value.
Without correction, the A260/A280 ratio of Sample 1
would be 1.52, a low value. When corrected by taking
into account the turbidity (A260-A320)/(A280-A320),
the ratio is 1.88, which is a more precise assessment

MK-AN-017 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

Figure 1: Absorbance spectrum of uncorrected and normalized
Sample 1 DNA.

In our experience, very few samples have a A260/A280
ratio of <1.6. In order to simulate such a sample,
Sample 1 was collected immediately after the donor
ate lunch and Sample 2 was collected immediately
after the donor drank coffee. The uncorrected A260/
A280 ratios of the two samples were 1.52 and 1.45,
respectively. When the A320 values were subtracted,
the corrected A260/A280 ratios were 1.88 and 1.80,
respectively (Table 1).
Example:Sample 1
A260 = 0.877; A280 = 0.576; A320 = 0.234		
Not corrected A260/A280 ratio =A260 ÷ A280 = 0.877 ÷ 0.576 = 1.52
Corrected A260/A280 ratio =(A260-A320) ÷ (A280-A320) = (0.877-0.234) ÷(0.576-0.234) = 0.643 ÷ 0.342 = 1.88

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2Methods to decrease the amount of turbidmaterial in purified DNA samples
As discussed, subtracting the A320 value from A260
and A280 will result in a more accurate A260/A280
ratio. However, some users may wish to remove as
much of the turbid material as possible. The following
describes approaches that can be used after standard
purification to clarify the samples further.

1. Additional centrifugation
Following purification and proper re-hydration of theDNA in TE or similar buffer, if a sample appears to beturbid, the user can centrifuge the sample for up to15 minutes at maximum speed. The DNA (still insolution in the supernatant) can be transferred to a
fresh tube, carefully avoiding contamination with thematerial in the pellet. The turbid material is composedof very fine particulate or colloidal material, so theamount removed will depend upon the speed andlength of centrifugation. In our experiments, we foundthat a 5 minute centrifugation at maximum speed(13,000 rpm, in a micro-centrifuge) was sufficient toremove about ½ of the turbid material (reduction ofabout 50% of the A320 reading) (Figures 2 and 3).
Extending the length of centrifugation to 15 minutes
removed a small amount more of the turbid material.
The additional centrifugation step did not affect theDNA concentration but did improve the uncorrected
A260/A280 ratio (Table 1).

2. Chloroform treatment
A small amount of chloroform added to the re-hydrated
DNA was found to efficiently and quickly remove
nearly all particulates that scatter light at 320 nm(Figures 2 and 3). To remove the turbid material, weadded 1/20th volume of chloroform (but no less than
2.5 µL) to the DNA dissolved in TE and vortexed thesample vigorously for a few seconds. Chloroform hasvery little solubility in water. The “coagulated” turbidmaterial and residual chloroform were removed by
a 10 minute centrifugation step at maximum speed.
The upper aqueous layer (containing the DNA) wastransferred to a fresh tube. DNA is not precipitatedby chloroform treatment.

Figure 3: Absorbance spectrums of purified DNA from Sample 2.

Care should be taken not to disturb the chloroform
layer or the pellet. When small amounts ofchloroform are used (e.g., 2.5 µL), the chloroform
phase may not be seen; only a small pellet ofimpurities may be visible.

Figure 2: Absorbance spectrums of purified DNA from Sample 1.

MK-AN-017 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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3Sample 1Sample 2

Treatments-5 min.
spin

15 min.
spin

CHCI3

-5 min.
spin

15 min.
spin

CHCI3

A260/A280

1.52

1.65

1.69

1.93

1.45

1.50

1.57

1.78

(A260-A320)/(A280-A320)

1.88

1.91

1.92

1.961.80

1.72

1.72

1.88

Concentration (ng/µL)

91.70

86.96

92.93

91.66

145.73140.45146.11

146.62

Table 1: Summary of A260/A280 ratios and DNA concentration for purified samples.

Despite the turbid nature of the samples described,
the purified DNA was both of high quality (asassessed by the corrected A260/A280 ratios, Table 1)
and of high molecular weight, >23kb (Figure 4A).
Furthermore, the treatments described did not haveany detrimental effect on the downstream processing
as assessed by PCR (Figure 4B).

Conclusions
In order to accurately evaluate the quality of eachpurified DNA sample, we strongly recommend that
the A320 value be subtracted prior to calculating
the A260/A280 ratio regardless of whether additional
purification steps were executed or if the samples
did not appear turbid to begin with. The turbidity
does not appear to affect DNA quality or downstream
processing. For users who wish to remove theseparticulates, we recommend first up to a 15 minutecentrifugation of the re-hydrated DNA samples. If aneven higher level of clarity of the samples is desired,
then a chloroform purification step of the rehydrated
DNA samples can be added.

Figure 4: Visualization of purified and PCR amplified DNA.
A: prepIT•L2P-purified DNA, Lanes 1,10: Lambda-HindIII marker;
B: 550 bp PCR amplicon of the human TS gene, Lanes 1,11: 1kb+ DNAladder; Lane 10: no template control; Lanes 2-9 in both images
correspond to samples as follows: Lane 2: Sample 1; Lane 3: Sample 1+ 5min spin; Lane 4: Sample 1 + 15min spin; Lane 5: Sample 1 + CHCl3;
Lane 6: Sample 2; Lane 7: Sample 2 + 5min spin; Lane 8: Sample 2 +15min spin; Lane 9: Sample 2 + CHCl3.

References
1Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.

Oragene®•DNA is not available for sale in the United States.
Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
®Oragene is a registered trademark and prepIT™ is a trademark 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.

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