My plasmid DNA sample is shown in white box on the picture below. It is almost unseen. Meanwhile, another bands is very clear to be seen altough their plasmid DNA concentration is lower than my plasmid DNA concentration (about 90 ng/microliter). Does anybody here can help me to explain this case? Thankyou so much for your attention.
Instead of nano drop, use Qubit fluorometer for accurate determination of DNA.
Has your DNA sample been purified? Degraded DNA also absorb light at 260 nm. The 260 nm OD indicated the amount of nucleotides in your sample, and not necessarily polymerized DNA. Purification kits allow removal of small size DNA and nucleotides.
Is your ratio 260/280 OK? For DNA it is usually between 1.8 and 2. If your ratio is lower, you might have protein contamination that increases the OD260.
Dear Emeline Vernhez, thankyou for your answer. Well my average ratio 260/280 is 1,986 according to nanodrop measurements (three times). It means that my DNA sample is pure enough right?
Yes this is a good ratio, so your sample does not seem to be contaminated by proteins.
Have you purified your sample to remove small fragments of DNA and nucleotides? The presence of degraded DNA is the only reason I imagine that could give a high OD260 but (almost) no band on a gel.
Other reasons could be during the loading process, some of the plasmid were not getting into the well properly or you forgot to put loading gel or sybr green.. hopefully it helps.
This could be is due to the presence of genomic DNA in plasmid DNA preparation
The problem with the NanoDrop is that is only capable of measuring nucleic acids, which is including DNA contamination and also free nucleotides, both of which you can exclude with qubit readings as you can go ds DNA binding after chromosomal dna degradation(with qubit) The Qubit promises to measure specifically either RNA or DNA and excludes free nucleotides
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