You will always get a bit of RNA with your DNA prep. In general, this is not a problem, since RNA is quickly degraded by RNases, that are abundantly present in most tissues. In your case, incubate your DNA samples with DNase-free RNaseA (which can be ordered from a vast number of companies), as already mentioned above. I used to resuspend my DNA pellet in 0.1mg/ml RNaseA in H2O. This immediately degrades RNA.

Hi Deepa, why are you sure that the bands is RNA?

How do you isolate the DNA? If you want to get rid of the RNA contamination, just use DNAse-free RNase. If RNase doesn't make the additional bands disappear, you'll know that it's not RNA...

Check the pH of the phenol, lower pHs (~4) are best for RNA, neutral or slight alkaline pH is best for DNA/RNA. Use fresh phenol, old phenol will oxidize and be less effective. Michael is correct, use an DNAse free RNase to degrade the RNA. Once you've optimized the nucleic acid isolation you can treat with RNase to isolate the DNA. If the bands disappear, you do indeed have RNA contamination.

@ Nadine A. Gund ...I run isolated RNA from same cell line with isolated DNA and found 3 bands in same position..

@ nathan Duval ..I tried other protocol also for DNA isolation , in that method also I got RNA bands .. and Phenol which I used was fresh and slight alkaline.

Which type of precipitation are u using?

@ celine Davoine ...Ethanol (first with 90 % than 70 % ethanol).

did u try with acetate sodium? Even if I doubt it will really be helpful. And yes u can get RNA when u isolate DNA. u can try a second phenol on your samples.

Using acetate sodium doesn't remove RNA; it precipitates all nucleic acids , I performed DNA and RNA extraction using phenol-chlorofom method so I think that you should use RNASE after heating it at 100 °C to eliminate DNASE to avoid degradation of your sample...Well I don't know if there is a kind of chemical compound to eliminate completely RNA contamination..

@Salma: The only thing I know which reliably kills RNAses is doing a Phenol-Chloroform extraction of the solution. RNAses are incredibly stable, to make them DNAse free you boil them for up to 30 minutes.

@Christian : I am speaking about boiling RNASE to remove contamination by DNASE and of course Phenol remove proteins/enzymes : RNASE or DNASE ect..autoclaving could kill RNASE or using DEPC..

You will always get a bit of RNA with your DNA prep. In general, this is not a problem, since RNA is quickly degraded by RNases, that are abundantly present in most tissues. In your case, incubate your DNA samples with DNase-free RNaseA (which can be ordered from a vast number of companies), as already mentioned above. I used to resuspend my DNA pellet in 0.1mg/ml RNaseA in H2O. This immediately degrades RNA.

@ deepa... please let me know how are you confirming those bands are of rRNA only??

yes it is possible to get RNA when isolating DNA, because both are nucleic acids and their extraction procedure is almost similar. One important thing, which I find worth mentioning in your case is that, for DNA isolation we use phenol with pH 8 and for RNA we use phenol with pH 4. The phenol which you are using is of pH 8 are you sure? Next option for you is to go for RNase treatment for removal of RNA impurities from DNA. Generally RNA should not contaminate DNA because RNases are more ubiquitous.

Good Luck

@Salma: You will not get all RNAse deactivated by autoclaving. Thats the reason people use DEPC. But besides that, the only really reliable way to get rid of them is Phenol/Chloroform.

@Deepa: When you do the extraction of DNA using alkaline Phenol (pH>7.8 is best for DNA Extraction) you will by default end up getting RNA molecules in the aqueous phase. Remember the principle: At pH>7.8, DNA will become highly soluble in water, but RNA molecules will get degraded partially by itself. At this pH, the electronegativity of the 2'-OH group of the ribose will become high and they will conduct a nucleophilic attack on the 5'->3' phosphodiester bond, which is the backbone of the RNA molecules. (Note: That's why you can't use this RNA any experiment). But unfortunately, this doesn't affect the solubility of the RNA in the water. So, when you run an agarose gel, you will find a blazing band at the bottom of the gel (Provided you don't run it too long!!!).

Now lets review what you have done. You said you did this experiment before but you didn't get these 3 bands and you confirmed that these bands are rRNA by running total RNA extract side by side. First of all, total RNA extract from human cell will show only two bands (28S and 18S rRNA) (Check this page for a pic: http://openi.nlm.nih.gov/detailedresult.php?img=2376114_87-6600419f1&req=4)

and mRNA smear. If you are very very lucky you can see a very faint tRNA band at the bottom of the gel. Check your observation note for the percentage of agarose that you used to run the genomic DNA and total RNA (RNA is run on formaldehyde denatured agarose gel). I bet that they aren't same. Rule is when you have run two different gels you have to compare the Rf (Retention factor) value of the bands in question and not the similarity in the pattern of running. Higher the percentage of agarose lower will be the mobility. So they are not the same and when you run your RNA contaminated DNA sample, you will only get very bright (Blazing) RNA smear at the bottom (Do another DNA extraction, don't give RNase A treatment and run a 0.7% prestained gel for about 15 to 30 min at 60V along with another properly RNase A treated sample and see for yourself). So, we have eliminated that the 3 bands are not RNA.

The other possibilities why you have 3 bands ( I am assuming that you saw some smear too in the gel) are

1. You have sheared your genomic DNA while extracting.

2. Exposure to non sterile environment combined with not properly keeping them on ice (Even though DNA are stable at higher temp, they aren't be stable in presence nucleases) will lead degradation, or

3. The cells that you used to extract the genomic DNA are dying.

Hope this answer helped you.

All the best for your experiment!

@Christian Praetorius: If you double autoclave the solutions, you will be able to get rid of RNase contamination. In our lab, we use double autoclaved double distilled water for all RNA biology work and till date, we haven't got RNA degradation even once.

@Ganeshkumar: Believe me, autoclaving doesn't solve all problems. Sometimes its the reason for them (e.g. if you prepacked sterile tips). Been there, seen that, got no T-shirt.

It's really helpful to read all the comments here from @Deepa's question, allow me to have a question here as it's very related to my current doubt. 

May I know what's the concept behind in detecting RNA contamination using A260/280 in isolated DNA?

(Note that the purity range for DNA is 1.80-2.0)

Thanks!