Can Southern blotting resolve 13.7kb and 11.5kb bands? - Help! (Feb/13/2006 )
I posted this on another message board, but I haven't gotten any replies. I'm kinda desperate for an answer, so please answer!!!
OK, I'm trying to detect one allele that is 13.7kb and another that is 11.5kb with probe A (basically trying to detect mutant and wild type bands in heterozygote cells).
Do you think I can successfully resolve 13.7kb and 11.5kb on an agarose gel? I think it's really hard.
Separating 4kb and 2kb is easy. Just use a 1% agarose gel and transfer to Southern blotting membrane.
However, 13.7kb and 11.5kb is hard cuz it's so high molecular weight. I'm thinking I should use a 0.5% agarose gel and run the gel real long before transfering. What do you think?
Also, some protocols say that agarose gel electrophoresis should be run very slow when doing Southerns. Like maybe running a gel 8hrs and then transferring. Is this necessary? And what is the purpose of running so slowly? Thanks
I use a different application (I blot for EMSA), but I use agarose gels and I too like to get nice sharp bands.
I use a 20x20 gel, choose your % based on band size (I think if I were you I would run 0.7 but that's your choice), and run it slowly over many hours. I typically run about 5-6 hours for blotting applications to get good resolution of the upper bands. I know it's 'old-school' and time-consuming, but i would rather not have to repeat the whole thing to get a scan-able blot if the data is good.
Another thought: use the conditions that allow you to run a gel to visualize with EtBR, that give resolution for those band sizes. do a trial gel before you set up your blot, to make sure the conditions are appropriate
OK thanks. I guess I'll try run the gel like mad so as to separate the 13.7kb and 11.5kb bands as much as possible.