Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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Well DNA and RNA are both known to absorb at 260 UV range. The reading at spectrophotometes at 260 corresponds both to DNA and RNA. Whwn we treat the extract with RNAase, the RNA has to get chewd up in some time , then the reading will totally be oF DNA.
I am facing a problem that my isolated dNA after RNAase treatment also, gives a reading at 260/280, more than 2, where it should be less than 1.8.
What may be the cause, plz help. On gel also there is no trace of RNA. I am thoroughly confused
Well, actually the plant is highly phenolic but luckily I dont face problems with it, on gel I observe some shearing and the 260/280 reading is most confusing. I havent encountered in the literature, a problem of this type.
But, I will do it 2-3 times again and see if it continues to be there.
but, what can be the reason, any suggesstions?
If RNAase treatement is altering the absorbacy then it is most likely that the absorbancy is due to the inter-ribonucleotidal linkage, as its the thing that RNAase removes. If in some way those ribo nucleotides form bonds again, either by arranging themselves on DNA templets or just combining with each others. Some other chemical reaction/s might also be 'using' the incident radiation, so as to increase absorbacy...
Tell me what u think...
I'm not very familiar with this technique so bear with me. My understanding is that it is the nitrogenous bases in nucleotides that absorb 260 nm light and that RNase cleaved the phosphodiester bonds linking the sugars. So are the bonds responsible for absorbing the the light untouched by the digest? If this is the case, running a mini-prep of the digest to remove the RNA nucleosides should purify the DNA, which you can then take a reading of.
It is not correct to state that by treating the extract with RNAse “the reading will totally be of DNA.”
In fact, RNAses simply cleaves the phosphodiester linkages of RNA producing free ribonucleotides, which still absorb at 260 nm.
Thus, unless you remove these free ribonucleotides from the reaction mixture (e.g., by dialysis, or by precipitating the DNA), you don’t expect the absorbance to decrease – as a matter of fact, it could even increase, because the extinction coefficient of the free nucleotides is often higher than that of the same nucleotides embedded in the RNA polymer.
8 posts • Page 1 of 1
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