User manual

Section 7- Diagnostics and Troubleshooting
• Wavelength accuracy of the spectrophotometers
Although the absorbance of a nucleic acid at 260nm is generally on a plateau, the absorbance curve at 280 nm
is quite steeply sloped. A slight shift in wavelength accuracy will have a large effect on 260/280 ratios. For
example, a
+ 1 nm shift in wavelength accuracy will result in a +0.2 change in the 260/280 ratio. Since many
spectrophotometers claim a 1 nm accuracy specification, it is possible to see as much as a 0.4 difference in the
260/280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are both within
wavelength accuracy specification.
• Nucleotide mix in your sample
The five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios**. The following
represent the 260/280 ratios estimated for each nucleotide if measured independently:
Guanine: 1.15
Adenine: 4.50
Cytosine: 1.51
Uracil: 4.00
Thymine: 1.47
The resultant 260/280 ratio for the nucleic acid being studied will be approximately equal to the weighted
average of the 260/280 ratios for the four nucleotides present. It is important to note that the generally accepted
ratios of 1.8 and 2.0 for DNA and RNA are “rules of thumb.” The actual ratio will depend on the composition of
the nucleic acid.
Note: RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of
Thymine.
** Leninger, A. L. Biochemistry, 2nd ed., Worth Publishers, New York, 1975
Unusual Spectrum
A sample that is ragged or exhibits jagged “cuts” out of the spectrum but is an otherwise normal shape, may be the
result of detector saturation. This may be caused by the software selecting too high of an integration time due to a
dirty pedestal. Clean both the upper and lower pedestal measurement surfaces as previously described and restart
the software.
If the problem persists contact
Technical Support. Outside of the US and Canada, please contact your local
NanoDrop products distributor.
Contact Technical Support
If after referring to the above troubleshooting tips you are unable to resolve your problem, please contact
Technical
Support for assistance. Outside of the US and Canada, please contact your local NanoDrop products distributor.
Thermo Fisher Scientific
3411 Silverside Road
Bancroft Building, Suite 100
Wilmington, DE 19810 U.S.A.
Telephone: [phone]
Fax: [phone]
E-mail: [e-mail]
www.nanodrop.com
Please have the serial number of the instrument available. The number can be found on the bottom of the unit.
7-7