I've isolated a plasmid with an alkaline lysis protocol, added RNAse and then measured the DNA concentration with a nanodrop. The yield was an average of 1300-1800 ng/ul. Yet, when pipetting 2 ul in an agarose gel it does not show up at all. The positive control, a plasmid isolated earlier and known to give bands, did work. Why could I get this result?
If your positive control gave band but not your newly isolated plasmid on the same gel, it means that some thing must be wrong with the latest plasmid isolation protocol. Although the DNA concentration was fine, it could be the reading of degraded plasmid or contaminant (check the purity too).
Nanodrop will measure all nucleic acids and that includes single nucleotides. You may be measuring completely degraded dna and rna bases. With nanodrop it is also vital that the nano is zeroed with exactly the same buffer/water as your dna was dissolved in. try loading a higher volume but a clean up of the plasmid dna looks worthwhile
Unlike the nanodrop, the qubit will measure only dsdna bound to the fluorescent dye complex, and it will ignore contaminants.
Dear Christiaan,
When you isolate genetic material it is important to look at the ratio of the 260 and 280 wavelengths. For plasmids (DNA) the ideal value is 1.8. Below that value you have proteins contamination, above you have RNA in your sample. If your sample was contaminated with RNA you'd have a band on a gel, so this is probably proteins contamination or you failed to isolate your plasmid. I'd recommend to repeat the lysis process and use silica columns for a better DNA concentration and quality ; you also can check your buffers. Good luck!
Steve : would it not take a high concentration of RNA to show up as a band? I get faint or no bands when I load less than 100ng DNA in a well, using EtBr
Amy: It depends; Christian's genentic material concentration was very high, so it must have been contaminated with something. He didn't provide us with the information on how much he had loaded on a gel, but assuming that the RNase treatment was successful, there is a low chance of finding RNA in his sample.
Amy: It depends; Christian's genentic material concentration was very high, so it must have been contaminated with something. He didn't provide us with the information on how much he had loaded on a gel, but assuming that the RNase treatment was successful, there is a low chance of finding RNA in his sample.
Steve Liebich My 260/280 ratios were all around 1.8 for these samples. The 230/260 ratios however were around 1.5. I know that is too low, but I am not sure how that would explain my results. These ratios are all similar to the positive control.
Since this post we've repeated the plasmid isolation with new E. Coli and doubled the amount of E. Coli per protocol and got results this time. It may be that, as Paul Rutland mentioned, that it was mostly measuring single nucleotides. The samples, after isolations, had the same DNA concentration as before, roughly the same 260/280 and 230/260 ratios but did yield positive results.
Christiaan Kinderman My doubt is how far you ran the gel after loading? Because after loading shorter run i.e 0.5cm in 0.8% gel is sufficient for as to visible clear bands after which it gets diminished. Try in this aspect.
Thanks
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