Thermo Scientific Evolution 201 - Concentration; Direct UV Assays for DNA, RNA, Oligonucleotides and Protein Concentration; Pathlength Correction Factors

18
nanoCell
288 Evolution 200 Series User Guide Thermo Scientific
Pathlength Correction Factors
Direct UV Assays for DNA, RNA, Oligonucleotides and Protein Concentration
Direct UV Assays for DNA, RNA, Oligonucleotides and Protein Concentration
Assay Unit
Calculation Factor
1.0cm Pathlength
Calculation Factor
1.0mm Pathlength
nanoCell
Calculation Factor
0.2mm Pathlength
nanoCell
Reference
dsDNA ng/μL 50 500 2500 Maniatis, T; Fritch, E. F.; Sambrook, J.,
Molecular Cloning: A Laboratory Handbook,
2nd Edition, Cold Spring Harbor Laboratory
Press, Plainview, NY 1989, Vol. 3.
ssDNA ng/μL 37 370 3650
RNA ng/μL 40 400 2000
Oligos ng/μL 33 330 1650
Protein 280 nm mg/mL 1 10 50 Fasman, D. G., Ed. Practical Handbook of
Biochemistry and Molecular Biology, CRC
Press, Boston 1992.
Stoscheck, C. M., Methods Enzymol, 1990,
182, 50-69.
Protein 205 nm mg/mL 31 310 1550
Assay Unit Equation
Factors
1.0mm
Pathlength
Calculation
Factor
1.0mm
Pathlength
nanoCell
Calculation
Factor
0.2mm
Pathlength
nanoCell
References
260/280
DNA/
Protein
μg/mL DNA and Protein Concentrations determined using:
DNA conc = [(A
260
- A
ref
)f
1
- (A
230
- A
ref
)f
2
]
Protein conc = [(A
230
- A
ref
)f
3
- (A
260
- A
ref
)f
4
]
260:280 Ratio =
Where:
A
260
= absorption at 260 nm
A
280
= absorption at 280 nm
A
ref
= background absorption at 320 nm
f
1
= 62.9
f
2
= 36.0
f
3
= 1,552
f
4
= 757.3
f
1
= 629
f
2
= 360
f
3
= 15,520
f
4
= 7,573
f
1
= 3,145
f
2
= 1,800
f
3
= 77,600
f
4
= 37,865
Warburg, O.;
Christian, W.
Biochem Z. 1942,
310, 384-421.
Groves, W. E.;
Davis, F. C., Jr.;
Sells, B. H., Anal.
Biochem. 1968, 22,
195-210.
260/230
DNA/
Protein
(Phenol)
μg/mL DNA and Protein Concentrations determined using:
DNA conc = [(A
260
- A
ref
)f
1
- (A
230
- A
ref
)f
2
]
Protein conc = [(A
230
- A
ref
)f
3
- (A
260
- A
ref
)f
4
]
260:230 Ratio =
Where:
A
260
= absorption at 260 nm
A
230
= absorption at 230 nm
A
ref
= background absorption at 320 nm
f
1
= 49.1
f
2
= 3.48
f
3
= 183
f
4
= 75.8
f
1
= 491
f
2
= 34.8
f
3
= 1,830
f
4
= 758
f
1
= 12,300
f
2
= 870
f
3
= 45,750
f
4
= 18,950
Kalb, V. F., Jr.;
Bernlohr, R.W.,
Anal. Biochem.
1977, 82, 362-371.
Warburg-
Christian
mg/mL Protein concentration is determined using:
Protein conc = [(A
280
x f
1
) - (A
260
x f
2
)]
Where:
A
280
= absorption at 280 nm
A
260
= absorption at 260 nm
1.55
0.76
15.5
7.6
3,875
1,900
Warburg, O.;
Christian, W.,
Biochem Z. 1942,
310, 384-421.
A
260
A
ref
–
A
280
A
ref
–
----------------------------
A
260
A
ref
–
A
230
A
ref
–
----------------------------