1DNA Assay Kit
Catalog # 6023
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify DNA

FORMAT:

96-well ELISA plate with non-removeable strips

ASSAY TYPE:

Dye-based assay/Fluorescence-based assay

ASSAY TIME:<0.5 hour

STANDARD RANGE:

10 µg/ml to 0.16 µg/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Solubilized tissue & cell culture samples (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

Varies (Typical DNA levels are usually 0.3-5 µg/mg wet tissue and 1-5 µg/1x106 cells)

FLUORESCENCE:

Excitation 360 nm/Emission 460 nm

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (1.7-7.6%)/Inter-Assay (5.2-8.0%)/Spiking Test (105.1-116.2%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6023 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2DNA Assay Kit
Catalog # 6023
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTIONDeoxyribonucleic acid (DNA) is a distinct cell component, therefore DNA amount is correlated with cell number (1). For example, in cartilage
tissue engineering, artificial cartilage quality is evaluated by DNA amounts translated as chondrocyte numbers, as well as amounts ofcollagen and glycosaminoglycans (GAGs) in extracellular matrix components (ECM) (2, 3). DNA can be quantified by the ratio of absorbanceat 260 nm and 280 nm, but this method is affected by contaminating proteins, RNA, and chemicals in the sample, requiring additional DNA
isolation steps. For tissue analysis, Chondrex, Inc. provides an alternative DNA assay kit employing the Hoechst 33258 fluorescent dyewhich specifically binds to Adenine-Thymine base pairs, resulting in fluorescence at excitation 360 nm/emission 460 nm. Because the dyeDNA binding and the fluorescence intensity are unaffected by contaminating proteins and other substances in an optimized assay condition,
this DNA assay kit works accurately with samples containing other analytes.
In order to facilitate further studies in this field, Chondrex, Inc. also provides various native collagen detection ELISAs, a Hydroxyproline
Assay Kit (Cat # 6017), Sirius Red/ Fast Green Staining Kit (Cat # 9046 ), Sirius Red Total Collagen Detection Kit (Cat # 9062), and GAGs
assay kit (Cat # 6022). Please visit www.chondrex.com or contact support@chondrex.com for more information.

KIT COMPONENTS
Item
Calf Thymus DNA Standard (60231)
Reaction Solution (60232)
Detection Solution Concentrate 200X (60233)
Black ELISA Plate

Quantity
1 vial
1 bottle
1 vial1 eachAmount100 µg/ml, 100 µl
50 ml
50 µl
96-well

Storage
-20°C
-20°C
-20°C
-20°C

ASSAY OUTLINE
Add 50 µl of diluted Standards and Samples

Add 50 µl of diluted Detection Solution to all wells

Incubate at room temperature for 5 minutes.

Read plate at Ex 360 nm/Em 460 nm© 2020 Chondrex, Inc. All Rights Reserved, 6023 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

PLATE MAPPING
123456789101112A

1010S1S1S9S9S17S17S25

S25

S33

S33

B

55S2S2S10S10S18S18S26

S26

S34

S34

C

2.52.5

S3S3

S11

S11

S19

S19

S27

S27

S35

S35

D

1.25

1.25

S4S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.63

0.63

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.31

0.31

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.16

0.16

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing
the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.

Table 1 - Solubilizing Reagents in the DNA Assay Kit
ReagentsGuanidine
SDS
Tween 20
Triton X - 100

Concentration in Samples
0.5 M
0.025%
0.0025%0.025%© 2020 Chondrex, Inc. All Rights Reserved, 6023 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: Take 40 µl of DNA Standard Solution and mix with 360 µl of Reaction Solution (10 µg/ml). Then seriallydilute it with Reaction Solution. For example, mix 200 µl of the standard (10 µg/ml) with an equal volume of Reaction Solution to makea 5 µg/ml solution, and then repeat it five more times for 2.5, 1.3, 0.63 0.31, and 0.16 µg/ml solutions. Chondrex, Inc. recommends
making fresh serial dilutions for each assay.
200 ml40ml

100 mg/ml

200 ml200 ml200 ml200 ml200 ml12345678360 ml

200 ml200 ml200 ml200 ml200 ml200 ml200 ml10 mg/ml

5 mg/ml

2.5 mg/ml

1.25 mg/ml

0.63 mg/ml

0.31 mg/ml

0.16 mg/ml

Blank

2.

Prepare Sample Dilutions: Dilute samples with Reaction Solution. The optimal sample dilution depends on the sample and preparation
methods; therefore, Chondrex, Inc. recommends multiple dilutions as a preliminary study.

3.

Prepare 1X Detection Solution: Mix 5 µl of 200X Concentrated Detection Solution and 1 ml Reaction Solution for each well. For
example, 8 samples, 7-point standards, one blank (all in duplicate) will require 1.6 ml of the 1X Detection Solution. Mix 8 µl of 200X
Concentrated Detection Solution and 1.6 ml Reaction Solution.
NOTE: Prepare the 1X solution just before use. Do not store and reuse the mixed solution for the next assay.

4.

Add Standards and Samples: Add 50 µl of diluted standards and samples into wells.

5.

Add 1X Detection Solution: Add 50 µl of 1X detection solution into all wells, and then incubate at room temperature for 5 minutes.

6.

Read plate: Read the plate at excitation 360 nm/emission 460 nm.

CALCULATING RESULTS
1.

Average the duplicate fluorescence intensity (FI) values for the blank, standards, and test samples.

2.

Subtract the averaged “blank” (B) FI values from the averaged FI values of standards and test samples.

3.

Plot the FI values of the standards against the DNA concentration (µg/ml). Using a log/log plot will linearize the data. Figure 1 shows
an example of a DNA standard curve from 0.16-10 µg/ml.

4.

The μg/ml of DNA in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain theDNA concentration (μg/ml) in the original sample.
NOTE 1: DNA levels in samples depend on the sample solubilization efficiency of individual protocols consequently, converting DNA
levels to cell numbers is not always applicable. Therefore, DNA levels as correlating to cell numbers can be used to compare samples
including positive and negative controls.
NOTE 2: To correlate DNA amounts to cell numbers, validating your DNA solubilization protocol is required. Please refer to thereferences for more information.

© 2020 Chondrex, Inc. All Rights Reserved, 6023 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

5Figure 1 - A Typical Standard Curve for the DNA Assay100000Fluoresence Intensity

R² = 0.99925

10000

1000

1000.1

110DNA Concentration (mg/ml)

ASSAY VALIDATION
Table 2 - Reproducibility Data for the DNA Assay Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.31 μg/ml
7.6
7.3
115.1%

0.13 μg/ml
3.6
5.2
116.2%

5 μg/ml
1.7
8.0
105.1%* Known amounts of DNA were added to samples and then diluted with Reaction Solution for assaying DNA.

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

V. M. Quent, D. Loessner, T. Friis, J. C. Reichert, D. W. Hutmacher, Discrepancies between metabolic activity and DNA content as toolto assess cell proliferation in cancer research. J Cell Mol Med 14, 1003-1013 (2010).

2.

J. H. Yoon, J. Halper, Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 5, 22-34 (2005).

3.

J. A. Buckwalter, H. J. Mankin, Articular cartilage: tissue design and chondrocyte-matrix interactions. Instr Course Lect 47, 477-486(1998).

© 2020 Chondrex, Inc. All Rights Reserved, 6023 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com