Hi Sneha

It looks as if...in your comparative results (with or without carrier RNA) the carrier RNA is interfering with your sample quality/preparation.....After elution you can remove it by using RNAse enzyme if the RNA is not desired....although the carrier molecules (tRNA, LPA and Glycogen) do not affect most of the downstream molecular biology applications...

Is the use of carrier RNA recommended by the kit?...these molecules are generally used when the concentration of nucleic acids are very low or they are small like (small RNA fraction) and regular purification protocols give less yield

since your yield is quite OK even without the carrier RNA....so you can isolate the viral DNA even without it...

and finally To rule out interference of carrier RNA in your sample OD values check quality of your samples on a gel based analysis....

bye

Hi Sneha

It looks as if...in your comparative results (with or without carrier RNA) the carrier RNA is interfering with your sample quality/preparation.....After elution you can remove it by using RNAse enzyme if the RNA is not desired....although the carrier molecules (tRNA, LPA and Glycogen) do not affect most of the downstream molecular biology applications...

Is the use of carrier RNA recommended by the kit?...these molecules are generally used when the concentration of nucleic acids are very low or they are small like (small RNA fraction) and regular purification protocols give less yield

since your yield is quite OK even without the carrier RNA....so you can isolate the viral DNA even without it...

and finally To rule out interference of carrier RNA in your sample OD values check quality of your samples on a gel based analysis....

bye

I think that a ratio=3 is not possible with just purin and pirimidine (d)oligonucleotides. Set 1 should contain something else to increase A260.

Hi Sneha.

Quote:

"Set 1 using carrier RNA molecule, which gave an A260/280 ratio of 3. DNA conc ~ 11 µg

Set 2 without the RNA carrier giving A260/280 ratio of 1.86. DNA conc ~ 0.6 µg"

What do you mean by "concentration of 11µg"? Per µl? If this is the case, dilute your sample around 100-fold to get concentrations of about 110ng/µl. The NanoDrop is much more accurate on that. The ratio you got migth be due to overload.

According to NanoDrop UserManual and Trouble Shoot, pure DNA would yield a A260/280 of 2.0, whereas pure RNA gives you 2.2.

Thus, a ratio of 3 must be due to something else, as Francisco said.

:)

Hi Sneha,

As previously said It is not possible to get ratio higher than 2 (pure RNA or DNA). Another possible mistake have been able to happen with the buffer that you have chose to do the blank. When i do this I check that the measure of the blank buffer after doing the baseline gives a plane line along the spectrum. Ideally your blank might be the same solution of your sample but without the DNA or RNA. Have you done the comparison with itheir respective blank buffer

Good luck

Carlos

@Carlos: I did use the same solution as blank that was used to elute the DNA. I took three readings each and blanked it twice. Though i'll try again with a different and see if the readings change. Thank you.

@Ulrike: Sorry for the incomplete data. The concentration is 11µg/150µl. :) Thank you

Hi Sneha,

as said before, for a pure DNA(RNA Sample the A260/280 ration should be between 1.7-1.9. However, if you are using columns for purification there are very frquently; wee are finding it regularly when using qiagen columns, but it does not interfere with our applications, therefore we are not too worried). The high arrparent yield with Set one most likely results from carrier RNA "contamination" (the membrane will bind all kinds of nucleic acids (RNA and DNA) although the affinity might differ). By photometric measurements however, you cannot discriminate.

You will have to find a different method to determine the viral DNA from the rest (maybe just a simple agarose gel electrophoresis should do the job.....?

Good luck

Volker

Hi sneha,

the 206/280 ratio for RNA or DNA is around 2, sometimes when the sample is highly diluted the ratio tend to get out of range. you can check that by preparing different dilutions and then do the reading. the last and the most convincing evidence that your DNA or RNA sample is good is by checking the gel. good luck

Agree with Lahoucine. Usely, I dilute too highly concentrated samples and the ratio is 'magically' good....

I do not agree with the carrier explanation. I will test it independently of RNA/DNA but it does not seems to be THE explanation.... Sorry avwioroko but I guess you are wrong...