The ratio has no effect. How could it?
A lower ratio indicates that the DNA sample does not only contain DNA (or, more precisely: nucleic acids) but also things that have an absorption maximum above 260 nm. Typical suspects are proteins (aromatic amino acids, especially phenylalanine, absorb maximally around 280 nm) and phenole (often residing from the extraction buffers). Protein contaminations are no problem. Often, the PCR works even better when some proteins are present. Many PCR mixes contain BSA (some protein mixture without enzymatic activities) to enhance the PCR. Phenol is the bad guy, since this inhibits most of the polymerases. The DNA sample should be free of contaminations of phenole (and other phenolic components). As far as I know, the OD 260/280 ratio is usually done to check for contaminating phenole.
If I removed proteins using a phenole-chlorophorm extraction, a low ratio likely indicates residing phenole that will casue problems in a subsequent PCR.
In contrast, if I have a phenole-free solution that may contain proteins, then this presumably has a low ratio, but this won't mean trouble for a subsequent PCR.
The ratio has no effect. How could it?
A lower ratio indicates that the DNA sample does not only contain DNA (or, more precisely: nucleic acids) but also things that have an absorption maximum above 260 nm. Typical suspects are proteins (aromatic amino acids, especially phenylalanine, absorb maximally around 280 nm) and phenole (often residing from the extraction buffers). Protein contaminations are no problem. Often, the PCR works even better when some proteins are present. Many PCR mixes contain BSA (some protein mixture without enzymatic activities) to enhance the PCR. Phenol is the bad guy, since this inhibits most of the polymerases. The DNA sample should be free of contaminations of phenole (and other phenolic components). As far as I know, the OD 260/280 ratio is usually done to check for contaminating phenole.
If I removed proteins using a phenole-chlorophorm extraction, a low ratio likely indicates residing phenole that will casue problems in a subsequent PCR.
In contrast, if I have a phenole-free solution that may contain proteins, then this presumably has a low ratio, but this won't mean trouble for a subsequent PCR.
Younes, have a look here:
http://www.biotek.com/resources/articles/micro-volume-purity-assessment-nucleid-acids.html
(Figure 7)
or
http://biomedicalgenomics.org/RNA_quality_control.html
(Figure 12)
It is correct that Trizol also absorbs at around 230 nm, so the 260/230 ratio can also be used to check for Trizol contamination. At 230 nm also GITC and components of the RLT tissue lysis buffer absorb.
Dear Inayat L,
The ratio you are asking tells the purity/Quality of DNA which you want to estimate, pure DNA will give value of 1.8 which can used for molecular analysis, for RAPD and SSR ratio value from 1.7 to 1.9 also work fine, make sure the conc. Of DNA is between 50-100ng/µl .
Attached is a Technical Bulletin (2 pages) from NanoDrop regarding the ratios of 260/280 and 260/230. At page 2, Figure 3 shows that two peaks were observed on the TRIzol (phenolic solution) run-- one at 230 nm and one at around 270 nm (near 280nm). The peak at 230 nm is much taller (more robust) than that one around 270 nm. So, probably both can be used to detect phenolic solution residuals, but 230 nm is a better one !?
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