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Spectrophotometer


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#1 theOwl

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Posted 03 February 2003 - 02:04 PM

What would be the best way to make sure that a spectrophotometer is accurately measuring the right absorbance for DNA? I have had the trouble that the concentration of a sample halved when measuring it in a different spectrophotometer.. both were recently checked by the company..

Would measuring different concentrations of free dNTPs be a good reference material?

#2 beslowboy

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Posted 10 October 2003 - 08:33 AM

i am also having similar problems. have you come up with an answer for you question?

#3 tfitzwater

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Posted 17 February 2004 - 05:17 PM

Ocean Optics http://www.oceanopti...bsstandards.asp
sells a kit containing NIST-Traceable Absorbance Standards (Catalog # STANABSUV) for $335. For measuring nucleic acids and proteins, the UV kit should be used. Care should be taken to avoid contamination of the solutions, as you will not get the correct numbers once contamination occurs. Prepare smaller aliquots and use those for routine use.

From their website: The two calibration standards kits (STAN-ABS-UV for ultraviolet, and the STAN-ABS-VIS for visible wavelength ranges) each contain a background reference for low, medium, and high absorbance standards, which should read within +/- 5% of the absorbance data provided. The absorbance standards are ready-to-use; preparation time and potential dilution errors are eliminated. All standards are non-toxic, non-carcinogenic, and have a one-year shelf life.

The STAN-ABS' polymer-based standards are submicron, non-surface charged, solid spheres in matrixes of ultrapure water. The standards' small bead size, coupled with Brownian motion, ensures that the polymer spheres stay in a homogenous suspension.

The STAN-ABS-UV kit is certified from 200-450 nm while the STAN-ABS-VIS kit is certified from 400-900 nm. NIST-traceable certificates, user instructions and data charts are provided in each kit.

Key features of the Ocean Optics absorbance standard kits:

NIST-traceable NIST-traceable certificates accompany each kit; the
kit-to-kit lot variance is better than +/- 1%. Absorbance charts are supplied with data collected on NIST-calibrated instruments.
Stable The standards are comprised of microsphere polymer beads suspended in ultrapure water and will not settle out of solution.
Non-toxic, non-carcinogenic No special handling, storage or disposal precautions need to be taken with the Ocean Optics standards. Material safety data sheets are supplied with each kit.
Easy-to-use The Ocean Optics standards are poured directly from their
125-mL bottles without dilution or special sample preparation. The standards are not photosensitive and do not require refrigeration.
Cost-effective Available for only $300 each, the kits contain a volume of standards that can provide up to 100 calibrations. The standards have a
one-year shelf life.

Experience has shown that spectrophotometers are not espcially accurate. Many years ago I observed that determinations of plasmid DNA concentrations from a UV-vis spectrophotometer and a Hoeffer fluorimeter did not agree. Tests were performed on ten different very clean supercoiled plasmid DNA preps (DNAs were the same size) that were banded in CsCl/EtBr twice, phenol extracted and ethanol precipitated. The DNA preps were obtained over a period of several years and stored at -20C. One g of each DNA prep was linearized with Hind III (as determined by both methods) and the reactions were stopped and loaded on a 0.8% agarose gel. After ethidium bromide staining (using a protocol I optimized for complete staining and destaining), Polaroid Type 55 film was used to obtain a negative for densitometer analysis. All DNA preps were completely linearized. There was a 10-fold variation in band intensity when the concentration was determined by spectrophometry. Band intensities were equal when the concentration was obtained by the fluorimeter.




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