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?
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
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
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)