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RNA absorbs at 260?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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RNA absorbs at 260?

Postby angel » Sat Apr 22, 2006 7:57 am

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 :(
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Postby 2810712 » Wed Apr 26, 2006 11:48 am

check for any other impurity, do it again.

Best Wishes.



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Postby angel » Fri Apr 28, 2006 6:03 am

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.
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Postby LilKim » Fri Apr 28, 2006 3:04 pm

I had a similar problem last year ... and no one could figure it out. I kept on doing it... and somehow it just got better on it's own. Hopefully the same will happen for you!

good luck
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Postby angel » Mon May 01, 2006 6:33 am

LilKim wrote:I had a similar problem last year ... and no one could figure it out. I kept on doing it... and somehow it just got better on it's own. Hopefully the same will happen for you!

good luck
-kim

but, what can be the reason, any suggesstions?
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Postby 2810712 » Mon May 01, 2006 7:49 am

angel wrote: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 :(


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...

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Postby dae » Sun May 07, 2006 4:14 am

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.
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Free ribonucleotides still absorb at 260 nm

Postby razor4 » Wed May 17, 2006 5:04 pm

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.
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