During visualizing my genomic DNA on an agarose gel, I came across two distinct bands of low molecular weight(around 100-300 bp) hanging along with my genomic DNA. What could be the reason for getting these bands? After reviewing I understood that it is either a plasmid DNA or ds RNA. But it is hard to distinguish as both of them look similar on a gel.
Is there a method/experiment to know what it is?
Hi Jigar,
Without seeing a picture of what you describe, it sounds like RNA contamination. Plasmid DNA would not be that small on an agarose gel. You can add some RNase to your genomic DNA and then after awhile run on an agarose gel and see if it is gone.
Good Luck!
I agree with Tom Masi. It's likely to be tRNA, based on the size you observed.
RNase treatment is one thing you can try.
Or, you can stain your DNA sample with SYBR Green I which hardly binds RNA.
In most cases, we will have that bands. It is RNAs... Treat the samples with 0.5 microgram/ml of Rnase and incubate for 15 min to remove that bands from your samples.
DNA purification is one of the important thing before gel run. Someone who wants to separate DNA he/she should input pure DNA into gel. To eliminate contamination it should use some RNase, Protease which degrade RNA and protein.
I am aware of RNA contamination in DNA isolation and RNAse treatment to remove it. I perform DNA purification by puregene kit before doing any downstream experiments. i have attached an gel image for you all to see and conclude better. Sorry for not putting it before. You would see double bands in below genomic DNA. I want to know what are they because I have never come across such bands while perfoming DNA isolation of same samples.
Hi,
That picure really helps.
How did you know that these bands correspond to 100-300 bp?
For me, the doublet bands looks like plasmids, and perhaps much bigger than 300 bp.
The fuzzy band seen in some lanes would be RNA contamination.
Those doublet bands are not 100-300bp. More like several kilobases in size based on where they are running in relation to the genomic DNA and the RNA cloud at the bottom of the gel. You should run a DNA ladder on your gel to get a better estimation of the size of those bands.
What cells are you extracting genomic DNA from? And why are the doublets not appearing in all the lanes that contain genomic DNA (1st lane and the last 5 lanes on the gel?
I am extracting DNA from fungal mycelia and you couldn't see those double bands in the first lane because that is positive control(standard) and the last 5 lanes are DNA samples of those same strains which I had isolated before. I run a gel of these samples again with along with an 100bp ladder which runs upto 1kb. These bands were above the 1 Kb band of ladder.
-Run control without treatment
-Run RNase treated sample: takes care of RNA
-Run Plasmid-Safe DNase treated sample: Takes care of gDNA but leaves plasmid intact
Also, you can try to transform the "plasmid" in E.coli, streak an aliquot of the transfromation mix on plates with different antibiotics and see if you can get colonies on any of the plasmids. That would also allow you to "clone" and characterize the plasmid.
These bands sound like ribosomal RNA to me. Try treating your sample with RNase and see what happens.
Also - though check this - I think SYBR green binds only to DNA, so if you stain with SYBR-green instead of ethidium bromide, the RNA bands should disappear. The same may well apply to more of the modern gel staining dyes.
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