Simple gel question - (Mar/31/2010 )
I am extracting DNA from tissue (using RNA later and a column of washing and eluting steps)
How do i prove what i ahve extracted is DNA? i assume a gel? i use agarose gels when looking into RNA and i am used to observing the two ribosmal bands. what will the DNA sample look like? based on the fact that it should be linear as i warm it before i add it to my gel. should it be a single bright band? or several bands? i have ran a few gels and seem to be able to get both of these types when altering the amount of agarose?
Why cant i run RNA and DNA on the same gel?
I am assuming that you are trying to get genomic DNA since you said tissue. gDNA looks like a rather large smear of high bp on an agarose gel - the agarose percent doesn't make a difference either. On a regular DNA agarose gel the RNA will be a smear also but lower in bp. There is nothing to say that you cannot run RNA on a DNA gel, or together, the problem is that the RNA will not be resolved at all - you will see no distinct bands.
georgiadave on Mar 31 2010, 08:14 PM said:
Sometimes you have distinct RNA bands in lower bp range, I had it for example with insect tissue after gDNA extraction. As i didn't wanted them they where gone after adding some RNAse...
i repeated the gel on a low % agarose and got several more bands.
can this occur becasue of over loading the gel?
also can you see traces of rRNA bands within genomic samples?
thanks for the answers i really appreciate it!
A lower % gel would allow the DNA to move more freely...I am not sure if this is the real cause for you seeing more bands but it seems plausible. You keep asking about the RNA but said you were extracting DNA - which are you interested in? RNA later protects the RNA, this is usually done for downstream applications that have nothing to do with the gDNA: rRNA banding, RT-PCR, etc... I guess I don't understand your question.
Sorry i am extracting RNA using an RNA column then washing DNA off the column i am getting high concentrations via a nano drop. intrestingly i am getting high 230nm with the DNA i assume this to be salt as i am using NaOH to wash the DNA off. i get good 260/280 ratios
I will attach a pic of my gel it has been run for 61mins (i stoped it at 41mins to have a look then placed in back in the tank for another 24mins) 1% agarose gel 80mV.
the first 2 lanes contain DNA at a high concentration and then the next two lanes are at a lower conc this is the same till the marker
lanes 10 and 11 contrain RNA i assume 1 of the rRNA bands has run off. the last 4 lanes are further DNA samples.
Below are the same samples as above but i used a higher amount of sample to gel loading buffer
9 - Marker
I think i need to treat the DNA with RNase and the RNA with DNase.
I was wonderign what is that stuff at the top of the wells ?
H = high conc of DNA
L= low conc of DNA
The genomic DNA in lanes 1-8 look good. Lane 5 has a very high concentration of DNA therefore it looks like it was somebody put their finger in silly putty - a small smear. The 'stuff' at the top of the wells looks to be DNA that is too large to move through the gel...if you look at lane 8 there isn't as much at the well but a faint band is visible. In my experience I have devised these two rules for gDNA....1) if the smear is above the band the DNA is degraded 2) if the smear is below the band the well is overloaded.
Is the marker 1KB ladder - from who?
The RNA has the same band as the DNA meaning you have DNA contamination in your RNA sample - use DNase.
The last four lanes look the same as the RNA lanes, not the DNA lanes...did you use a different protocol? You are using an all-in-one kit? If so you may need to take more care with the elution steps, use a second RNA elution after the first to remove any that is left, or find a kit that allows you to remove the DNA first and use an on-column DNase digest.
Another option is using a two column kit, these are available from many suppliers. Add lysate to first column, spin, add 1 volume 70% EtOH to the flow-through and load onto another column. The first column will bind DNA and the second will bind the RNA. This would clean up your samples.