What is the exact protocol for quantification of DNA using UV visible spectrophotometer?
Dear Sir. Concerning your issue about the exact protocol for quantification of DNA using UV visible spectrophotometer.
If sample is pure (i.e. without significant amounts of contaminantes suchas a proteins, phenol, agarose, or other nucleic acids), can use spec to measure amount of UV irradiation absorbed by the bases.
For quantitating DNA or RNA, readings should be taken at wavelengths of 260 nm and 280 nm. The reading at 260 nm allows calculation of the concentration of nucleic acid in the sample.
1 O.D. at 260 nm for double-stranded DNA = 50 ng/ul of dsDNA
1 O.D. at 260 nm for single-stranded DNA = 20-33 ng/ul of ssDNA
1 O.D. at 260 nm for RNA molecules = 40 ng/ul of RNA
The reading at 280 nm gives the amount of protein in the sample.
Pure preparations of DNA and RNA have OD260/OD280 values of 1.8 to 2.0, respectively. If there is contamination with protein or phenol, this ratio will be significantly less than the values given above, and accurate quantitation of the amount of nucleic acid will not be possible.
So typically, dilute sample 1 ul in 100 ul so the dilution factor is 100. Put whole 100 ul in spectrophotometer cuvette. The DNA concentration read will then be:
OD260 * 50 ng/ul * dilution factor
For example, if have OD260 = 1.6. Then the concentration is:
1.6 * 50 ng/ul * 100 = 8000 ng/ul or 8 ug/ul. I think the following below links and the attached file may help you in your analysis:
http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/Spectrophotometry.pdf
http://schepartzlab.yale.edu/intranet/protocols/Ultraviolet.pdf
http://www.ableweb.org/volumes/vol-22/5-clark.pdf
Thanks
Unchained Labs is having a webinar tomorrow (Sept. 12th) on DNA quantitation.
Dear Nisha,
Most of the modern day UV-VIS Spectrophotometer will have a standard method for the quantification of DNA/RNA. I am associated with Shimadzu and we offer this programme by default in our equipment.
The Implen NanoPhotometer has a great application method. It's very intuitive and straight forward.
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