RNA gel to test DIG labeled RNA probe - (Feb/16/2010 )
Wondering if anyone can help me with a wee problem....Im going to make a DIG labeled RNA probe and want to test the probe when finished. From my reading there seems to be two ways that you can test the probe, the most common is the DOT blot which I understand as there is plenty of information to be found on this protocol. The second way is to run the probe in an RNA gel. My problem is I cant find information on whether I should denature the probe before running it and what I should see in the gel after it is run.....essentially how do I know the integrity of the probe is good????
Thanks, any help would be greatly appreciated
The dot blot and the gel tell you two different things - The blot tells you if your probe binds (though not specificity) and potentially at what dilutions of probe. The gel tells you if you have a probe at all, i.e. you are looking for a band of the correct size. Run next to an unlabelled probe if you can, the DIG retards the migration of the probe through the gel so will appear larger in MW than the unlabelled.
You should denature your probe before running.