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southern hyb solution
Posted by: nathan (IP Hidden, New member, 4)
Date: May 29, 2005 08:44PM
hi all,
just wondering what people are using as their hyb solution? i'm trying to get my southerns to work- the only other thing that i can think of to alter is the hybridisation. i get good DNA digestion, good transfer (checked on the gel and filter), i know the porbe works as it gives good signal on a northern. any other tricks i can try? thanks, nathan
Re: southern hyb solution
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 30, 2005 08:59PM
Hyb solution usually has optimized composition (from text book or the one of commercially available), but if you would like to play with it, SSC and salt conc. will be 2 key components to begin. Hyb temperature will be next factor to test followed by washing condition - stringent or relaxed....BTW, Southern is different from Northern, double stranded DNA vs. single-strnded RNA so denaturation of your DNA will be another thing may go wrong.
Re: southern hyb solution
Posted by: nathan (IP Hidden, New member, 4)
Date: June 21, 2005 11:06PM
thanks for the tips.
i have now tried a hyb solution with formamide, dendharts and dextran sulphate- gives me a signal but too much back ground! can i reuse this probe? its in hyb at -20C- how do i denature it before putting onto to a new blot? any help would be greatly appreciated. thanks, nathan
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