Parameters for Southern Hybs!

[Gayatri Mani]

Dear scientists,

I am having a lot of trouble with southern hybs of genomic DNA from CHO 
cells.The fragments I am trying to resolve are 2000,2500,5000,7500bp 
long.The 2500bp and 5000bp fragments resolve however not the higher 
molecular weight fragment.In my gel washing step,I depurinate the gel by 
immersing it for 20 minutes or so,in a solution of 0.25 HCl. would that be 
too long or too short a time and could that be the reason why my bands are 
coming up? Also,I am having to expose the film for at least 10-12 day before 
any of the CHo clones come up.Could the time of exposure be shortened in any 
way, say if I didn't add formamide to my hyb mix or if i used intensifying 
screens.Any suggestions would be great.
Thanks .

Gayatri


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