Help needed for removing RNA in DNA extract. - (Sep/06/2005 )
I was trying to extract a nice phage DNA for my own use in some digestions and ligations analysis. However, I can't get rid of the RNA in my extract no matter how much I added in the RNAse A and how long I incubated it. For the information, I used as much as 1mg of RNaseA for a total reaction volume of 1ml with incubation at 37C for as long as 2 hours. Unfortunately, the RNA keep showing itself. I had also tried doing twice the phenol chloroform extraction and twice the RNase A treatment but they can't help.
The gel photos showed no intact RNA as smearing was observed at the bottom. Therefore, I think that thw RNA is not chewed to pieces small enough to be extract out during phenol chloroform extraction?
Can someone please help? Really appreciate it.
Thank you so much.
Are you not just looking at contamination with genomic DNA?
It's quite unlikely for it to be the genomic DNA since there is no smearing down from the phage DNA band. I treated my samples with DNase I and get rid of it using EDTA with SDS before breaking the phage coat. What I had observed was one upper band one shadow at the bottom.
Thanks for your reply Theo22, really appreciate it.
RNase doesn't digest double strandard RNA and only cleaves between purines (I think). If you want complete digestion then you will need to add RNase T1 as well.
Better DNA sequencing
First of all, thanks a lot for replying. I have the DNA band on the top of the gel lane followed by a smearing at the bottom of it, which indicates most likely as degraded RNA. The problem I have now is that I can't remove it completely.
With this, I had attached a gel photo for reference.
Thank you so much for your helps.
The photo is not much help as you don't have any labels or markers. Can you upload a better photo?
PCR sequencing DNA
I just had a look at your gel. The fuzzy band at the bottom looks like e.g ficoll orange from your loading dye. Which kind of loading dye are you using?
Here's another gel photo, hope this is good enough. I can't get rid of the degraded RNA even with twice of phenol chloroform extraction.
Thank you so much for helping.
M = Marker of phage DNA cut with HindIII
Lane 1 = sample of DNA extract with degraded RNA
By the way, the loading dye, which I am using is 6X loading dye that contains, 0.09% bromophenol blue, 0.09% xylene cyanol FF, glycerol, and EDTA.
Just check whether your loading dye is not running exactly at the same spot as your 'RNA'. I wouldn't be surprised....