southern blots - (Apr/15/2004 )
Hi can anyone answer this
I am having problems with my southern hybridization.
I am wondering if the DNA transfers to my membrane.
Is it possible that the DNA can wash into the solutions when denaturing and neutralizing?
Try running a control blot with plenty of DNA (salmon sperm or other) cut with random enzymes. View the nictrocellulose filter after every step if DNA can be seen-if it is, your blots are just fine as far as transfer issues are concerned.
Poor signals on a Southern are usually due to probe defects coupled with incorrect hybridization temperatures-rarely due to DNA washing away unless the filter is old, brittle or exposed to too much light in storage or to ammonia/HCl from a nearby chemicals closet. Useful results have also been achieved when you do the primer hybridization step without blocking and/or running a primer hyb at 25C. This way you will get a lot of noise with the signal and then noise can be cleared away by increasing temperature or other variations.