DNA quantification of Oragene®/saliva samples† using SYBR® Green I Dye
and a micro-plate reader
Introduction
Measurement of absorbance at 260 nm (A260) is commonly used for quantifying DNA. Disadvantages ofusing A260 include (i) insensitivity of the assay, and (ii) interference by non-DNA components such as RNA,
particularly in samples that are not highly purified. This assay, developed by DNA Genotek, uses SYBR Green I,
a fluorescent dye which has specificity for double-stranded (ds)DNA. The purpose of this protocol is to quantify
purified total dsDNA by fluorescence.

Equipment and reagents•

Micro-plate reader (e.g., TECAN – M200 96-well plate reader)•96-well black plate (e.g., Greiner – 96-well plate, black. VWR Cat. No. 82050-784)•1x TE (10 mM Tris HCI, 1 mM EDTA, pH 8)––Store at room temperature•

SYBR Green I dye (Invitrogen Cat. No. S7563)––100x working stock prepared by diluting 10 µL of SYBR Green I dye with 990 µL TE buffer––Store in 10 or 20 µL aliquots in 0.2 mL PCR tubes at -20°C––Before each use, thaw at room temperature. Discard unused portion•dsDNA for Standard Curve – Lambda DNA (Invitrogen Cat. No. 25250-010)––Serially dilute the DNA to give a total of 7 dilutions plus a no-DNA point––50 µL aliquots of each standard are stored in 0.2 mL PCR tubes at - 20ºC (See Table 2. Standards A-H)––Thaw one tube of each standardProcedure
1. Preparation of master mix
Prepare a master mix solution, sufficient for all tubes to be assayed.
Volume(µL)
TE94100x SYBR Green 1

1Total95

20 + nn = number of unknown samples

Table 1†

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

(outside the USA)(in the USA)

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2. Standard curve
For each standard (A-H):
a. In duplicate, add 5 µL of each standard to a well of a black 96-well plate.
b. Add 95 µL of the master mix to each well.
Standard

Concentration(ng/µL)

Volume(µL)

Total DNA(ng)

A10.0

550.0

B

5.0

525.0

C

2.50512.5D

1.25

5

6.25

E

0.625

53.12

F

0.312551.56

G0.156

50.78

H0.0

50.0Table 2

3. Unknown purified saliva samples
For each unknown sample (n):a. Dilute purified DNA 1:50 in 1x TE (4 µL sample + 196 µL 1x TE).
b. Add 5 µL of unknown sample to a well of a black 96-well plate.
c. Add 95 µL of master mix.
4. Read fluorescence of samples
a. Excitation 485 nM.
b. Emission 535 nM.

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 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 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-075 Issue 3/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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