1. Understanding Variations in
Oligonucleotide OD and Yield
CHIA Jin Ngee
Integrated DNA Technologies

2. Terminology
 Scale is the starting amount of material
 nanomoles (nmol)
 Yield is the delivered amount of material
 nanomoles (nmol)
 OD units
 OD (optical density) is a measure of concentration

3. Areas of Confusion
 1 OD of single-stranded DNA = 33 µg/mL
 True for large single-stranded DNA 1 kb and longer
 Why does this not apply to short oligonucleotides?
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Each base exerts an influence on the overall absorbance
Each neighboring base exerts an influence on the overall absorbance
Each oligonucleotide is custom made and has a unique sequence
Therefore, what constitutes 1 OD is unique for each oligonucleotide

4. What is Absorbance or OD?


5. Molar Extinction Coefficient for Oligonucleotides
 Nearest-neighbor method
 For sequence: 5′–dATGCTTC–3′
 ƐdATGCTTC = ƐdApdT + ƐdTpdG + ƐdGpdC + ƐdCpdT + ƐdTpdT + ƐdTpdC - ƐpdT - ƐpdG - ƐpdC - ƐpdT
- ƐpdT
 Wavelength of 260 nm, neutral pH
 General constant extinction coefficient for single-stranded DNA
 33 ng-cm/µL*
 Use IDT OligoAnalyzer® program to determine the value of 1 OD
 http://sg.idtdna.com/analyzer/Applications/OligoAnalyzer/
*Some instruments use this constant extinction coefficient by default

6. Molar Extinction Coefficient for Bases
DNA
RNA
Stack or monomer
Extinction coefficient
Stack or monomer
Extinction coefficient
pdA
15400
pA
15400
pdC
7400
pC
7200
pdG
11500
pG
11500
pdT
8700
pU
9900
dApdA
27400
ApA
27400
dApdC
21200
AdC
21000
dApdG
25000
ApG
25000
dApdT
22800
ApU
24000
dCpdA
21200
CpA
21000
dCpdC
14600
CpC
14200
dCpdG
18000
CpG
17800
dCpdT
15200
CpU
16200
dGpdA
25200
GpA
25200
dGpdC
17600
GpC
17400
dGpdG
21600
GpG
21600
dGpdT
20000
GpU
21200
dTpdA
23400
UpA
24600
dTpdC
16200
UpC
17200
dTpdG
19000
UpG
20000
dTpdT
16800
UpU
19600

7. Sequence-Influenced OD Results
Name
GC+AT=Length
Sequence
Mol. wt
Ɛ
nmol/OD
µg/OD
50/50 mix
10+10=20
AGAGAGAGAGAGAGAGAGAG
6362.2
234700
4.26
27.11
50/50 separated
10+10=20
AAAAAAAAAAGGGGGGGGGG
6362.2
223900
4.47
28.42
Actin B F
10+10=20
TTGCTGACAGGATGCAGAAG
6206.1
200500
4.99
30.95
Actin B R
10+10=20
TGATCCACATCTGCTGGAAG
6117.0
190800
5.24
32.06
Tubb5 F
11+10=21
GATCGGTGCTAAGTTCTGGGA
6517.3
205900
4.86
31.65
Tubb5 R
11+10=21
AGGGACATACTTGCCACCTGT
6406.2
199300
5.02
32.14

8. Variation Due to Calculation Method Used
 Assume absorbance = 1.0 and path length = 1.0 cm
Nearest neighbor
Constant
Sequence (5′–3′)
AGAGAGAGAGAGAGAGAGAG
4.26
27.11
5.81
33.00
AAAAAAAAAAGGGGGGGGGG
4.47
28.42
5.81
33.00
TTGCTGACAGGATGCAGAAG
4.99
30.95
5.32
33.00
TGATCCACATCTGCTGGAAG
5.24
32.06
5.39
33.00
GATCGGTGCTAAGTTCTGGGA
4.86
31.65
5.06
33.00
AGGGACATACTTGCCACCTGT
5.02
32.14
5.15
33.00

9. OD Unit


10. Variation Due to Instruments
 A major source of variation
 IDT calibrates spectrophotometers regularly
 Variations between our measurements and customers’ are always
observed
 Despite using the same nearest neighbor method
 20–30% variation between IDT readings and yours

11. Yield
 Yield is calculated from OD
 Variation between your measured and calculated OD and ours
 Yield is based on OD; therefore, variations are due to the OD measurements
 For more details, see the article:
 “Oligo Quantification—Getting it Right” in DECODED 3.1 (January 2013 issue) at
www.idtdna.com/decoded

12. Conclusions
 OD variation will always exist between us and you due to
differences in instruments used
 Do not be alarmed at these variations
 What if your OD determinations reveal a very large variation?
 Contact us:
 asiacustcare@idtdna.com
 Chat with us through our webpage www.idtdna.com
 Call us at +65 6775 9187 (IDT Asia)

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