I isolated plasmid DNA by alkaline lysis method.I am getting a very weird graph in nanodrop. No peak was seen at 260 nm.Can anyone please tell what is the problem?
The problem you are having is that your sample has such high absorption in the UV region that you are beyond the range of the machine, hence the plateau between 200 and 300nm. So if this were real, you could just dilute your sample (probably 10-fold in this case) and repeat to see the peaks. However this result is quite surprising and unlikely to be plasmid. In theory it could be contamination by protein or other nucleic acid (like RNA or genomic DNA) but more likely it is contamination by some aromatic compound. Did you by chance phenol extract as part of your plasmid prep, even a little bit of phenol will give you absorption through the UV spectrum? If so, then you have not removed all the phenol.
Thank You sir.... Yes I used phenol as part of my plasmid prep.....I added phenol: chloroform :isoamyl alcohol and took the aqueous phase in a new tube ....then I added chloroform to that to remove phenol...After centrifuging , there wew two phases formed and I took the upper phase.Is this the correct method to remove phenol? Kindly provide your suggestions
normally you don’t need to do a phenol extraction after an alkaline lysis miniprep, but since you have already done it, try ethanol precipitating your DNA to remove the phenol.
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