I did agarose gel electrophoresis with different concentrations of Dna:250ng,500ng and 1 microgram.But the bands were in smear form,from which I concluded that the dna was degraded.But when i checked the purity of the Dna in a nanodrop,it was found to be 1.86.where did i went wrong?can somebody pls explain?
Hi Nisha Reghu
The amount of DNA you are loading for agarose gel electrophoresis is actually too high; so, you will always obtain a thick band with smear. Try to load lower amount (lower than 300 ng). If it is a smear, it does not mean that the DNA sample is degraded to a single nucleotide or very small fragment, but of different sizes of DNA (for degraded product, lower than the expected band). If you are using ethidium bromide or Midori green type stains to visualize DNA samples on gel, they bind to DNA by intercalating between DNA base pairs, so they can easily bind to smaller fragment of DNA, that's why you are getting higher value of A260/A280 ratio, which means your sample is significantly free from protein contamination. The purity of your DNA is acceptable (low protein content), but there can be degraded products. Agarose gel electrophoresis visualization and absorbance ratio: these two are independent to each other.
Best wishes.
Degradation of dna and purity of dna are completely independent of each other. Pure dna can be degraded into smaller fragments and dirty impure dna can be high molecular weight and not degraded, As your dna seems to be degraded you may need to look at the storage of the sample that the dna came from and possibly the process used to lyse the cells as most degradation happens when the cells are lysed and if the lysis buffer does not contain EDTA then nucleases that are also released during lysis will be able to degrade your dna
Hello Nisha Reghu
250ng and above is a lot of DNA. DNA of up to 100 ng per well will result in a sharp, clean band on an ethidium bromide-stained gel using a high range marker to visualize the integrity of DNA. If you are referring to genomic DNA which is larger in size, it will usually show up at the very top of your gel (0.8-1% agarose gel) i.e., very close to the well.
Before assuming that the DNA is degraded, I suggest you first try decreasing the amount of DNA to be loaded on the gel (use 100ng DNA). Also, improper electrophoresis conditions may be the cause. Do not allow voltage to exceed 20 V/cm and maintain a temperature of less than 30°C during electrophoresis. Do check that the electrophoresis buffer used has sufficient buffer capacity.
Even after trying the above, you fail, then something may have gone wrong during DNA extraction. DNA degradation may have happened during the extraction process and not during electrophoresis. For instance, DNase contamination or DNA handling issue like mechanical shearing or the presence of too much salt in the DNA for which you could use ethanol precipitation to remove excess salts prior to electrophoresis.
Hope these suggestions work!
Best.
Malcolm Nobre, Thank you for your answer, sir.
I'm using low EEO agarose and I'm running genomic DNA. Do you know if it has anything to do with the run?
- Badges
- Science method