Simple gel question
Posted 31 March 2010 - 10:01 AM
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?
Posted 31 March 2010 - 10:14 AM
Posted 31 March 2010 - 10:56 AM
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 01 April 2010 - 06:58 AM
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!
Posted 01 April 2010 - 08:57 AM
Posted 02 April 2010 - 03:46 AM
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 ?
Posted 02 April 2010 - 10:57 AM
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.