I extracted the DNA using modified CTAB method, the pallet colours were good and DNA was not sticky. I used 100 voltage for 35 minutes, when i got the result i am totally socked because two bands : one near the well which is not degredaded and another far from the well but degrated and also smearing was observed.
i am looking for suitable ideas which will help me to go forward
I'm honestly not shocked by the big band close to the well which moved into the gel at the migration limit: it is genomic DNA you're working on, it consists of big size DNA molecules. Plus, it seems you loaded quite a lot of material. I see the same thing when I check the absence of degradation in my genomic DNA samples. As for the lower smear you observe, have you treated your samples with RNase A? If not, it is most likely RNA you're looking at. Try to add RNase A to eliminate it if it bothers your downstream applications, but be sure the RNase A you're using is DNase-free beforehand.
It seems that your DNA is not completely resuspended. I got similar bands when I resuspended the genomic DNA pellet in too little buffer (for example, 20 microliters). After adding 30 microliters of buffer and careful resuspension separation of fragments was good.
Cheers, Christrian
I'm honestly not shocked by the big band close to the well which moved into the gel at the migration limit: it is genomic DNA you're working on, it consists of big size DNA molecules. Plus, it seems you loaded quite a lot of material. I see the same thing when I check the absence of degradation in my genomic DNA samples. As for the lower smear you observe, have you treated your samples with RNase A? If not, it is most likely RNA you're looking at. Try to add RNase A to eliminate it if it bothers your downstream applications, but be sure the RNase A you're using is DNase-free beforehand.
What remains near the well is junk, sometimes protein or other contamination. And as Andrea said you have loaded quite a lot of DNA.
I do not discard a little degradation of your DNA. Try to dilute it and load it again and let us know what happened.
Deraded gDNA or plasmid display as a band and do not migrate form the wells.
You can just combine the answers from Andrea and Paramita, and you will got the most possible reasons. You should dilute you DNAs a bit as you definetely loaded too much DNAs, also I suggest you rerun the gel electrophoresis under a higher voltage, say 110, and shorter time, because DNAs may be degraded to some extent if you run for a long time.
That is not uncommon. As matter of fact, although a few samples you did not get good DNA, I think in general the turnout of your gDNA isolation from the other samples were pretty good...
Thank you all for your valuable ideas, I will update the result after I run the samples with dilution.
Dear Bal Kumari Oliy,
The DNA you have extracted was very high in concentration, so you have found a little bit smear in the area adjacent to the loading well. I think it not a band, it happen due to high concentrated DNA .
It's because of overloading the gel and the smear belov your genomic DNA is most probably RNA contamination. Do a RNAse Treatment of an aliquot of your DNA sample u will see it will not be there anymore. Actually the same like Andrea said.
you have good quantity of DNA, need to treat with RNAse for getting fine bands
The final DNA you have eluted into is highly concentrated and you have used too much of DNA to load in the well for checking. If you are checking for quality of DNA, a mere 20 ng of DNA can be visualised on the gel when stained with EtBr. Also, before the last step of dissolving the pellet, try to treat with RNAse to degrade the RNA you are getting along with DNA sothat you can use for further
Treat your DNA samples with RNase and load less amount of DNA sample in 0.8% agarose gel
This is fine. No reason to be shocked. Is it 1% gel? Some degradation is normal so do not worry. More than 95% genomic DNA that you have is intact and good for most of the downstream applications.Only thing I would say is your lanes are overloaded. If you run one third of the quantity per lane, your genomic DNA band will be sharper and degradation will also go down appreciably. I would not worry at this point.
Good luck.
Nirmal
It is quite normal if your samples are concentrated and the wells were overloaded. you can try to dilute the samples and run your gel at lower voltage and longer time. I recommend 90 V for 35 min on 1% agarose.
Similar results were observed by me too, during G DNA agarose gel electrophoresis..
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