I had a sample of plasmid extracted from e.coli and wanted to check the amount of Host cell RNA impurity with a fluorescent dye. to do this, first of all, I treated the sample with DNase I.
(NEB kit)
sample 80 μl (concentration: 25 μl/ml)
Buffer 10 μl
DNaseI 1 μl (2 unit)---> (1 unit DNaseI degrade 1 μg DNA in 10 minute)
H2O 9 μl
then I run agarose gel electrophoresis for two samples (DNaseI treated, untreated sample). surprisingly saw no differences between them and I don't know why?
(blue: untreated sample, pink: treated sample)
Any useful idea would be appreciated :)
I think the problem is this: according to https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011904_Dnase_I_RNasefree_UG.pdf, "DNase I degrades DNA by making random single-strand nicks in the phosphate backbone".
This means that DNAse I digestion is good for linear DNA fragments. When the DNA is circular, the fragments are likely to remain coiled together. To linearize the plasmid, two nicks should overlap, hence it is a rare event.
I suggest using some denaturing agents such as urea to facilitate the unfolding of the plasmid. Or, even better, linearize the plasmid with a specific primer before the DNAse I treatment.
Having said that, why are you digesting the plasmid to remove RNA? I would have used an RNA specific RNAse such as RNAse A.
Hello Fateme Nasiri
DNase I can degrade plasmid DNA. The activity of DNase I depends on the ionic strength of the reaction buffer. The enzyme has optimal activity in buffer containing Mg2+ and Ca2+. Micromolar levels of Ca2+ acts as an enzyme activator in the presence of Mg2+.
Does the buffer you use contain Mg 2+ and Ca2+ ?
Use a higher concentration of DNase I and incubate at 37°C for 10 mins.
Good Luck.
DNase hydrolyzes single-stranded DNA faster than double-stranded DNA. The supercoiled plasmid DNA "breathes" in solution due to the high tension of the double helix with closed ends. Supercoiled DNA is more compact due to supercoils; during electrophoresis in agarose gel, it moves quickly as a narrow band. Supercoiled DNA always contains "loops" of single-stranded DNA regions, which are rapidly hydrolyzed by DNase. It leads to the relaxation of the supercoiled DNA, and it converts into an open circular form. This DNA moves much more slowly in the gel. It is slowly hydrolyzed by DNase now, thus forming a linear double-stranded plasmid DNA, which is visible in the gel as a wider band. Its mobility depends on the size of the plasmid and the conditions of phoresis. For medium-sized plasmids, it has intermediate mobility between supercoiled and open circular forms of DNA.
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