Why my plasmid DNA gets degraded in Neb 10x buffer (2.1), used in restriction digestion? What can be the most probable reason for this?
Is it possible that you have isolated rna not plasmid dna and that is what is looking degraded. Do you have a picture of plasmid dna in water or elution buffer and plasmid in buffer 2.1. The buffer is not likely to degrade dna unless it has been at room temperature for a long time and has something growing in it that produces nucleases but a picture would be helpful
What is the host strain you isolated the plasmid from? If it is an EndA+ strain then your plasmid preps will have endonuclease contamination that gets activated when you add any restriction buffer containing Mg++
One effective way to circumvent this (other than using an EndA- strain) is to not treat your mini prep with RNase. The RNA will serve to inhibit the EndA activity.
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