Hi everyone. I'm doing right now some southern blotting assays on a plant genomic DNA and several questions have arised.
1. Why is it necesary to digest over night? I had seen a simmilar pattern between over night digestions and digestions of a few hours (2 or 3). But is it really determinant on succesfull of the reaction?
2. Why is it necessary a over night electrophoresis at low voltage? Again, at least for molecular weight marker, it is possible to obtain a good resolution appling a higer voltage (around 70V) for 2 hours. But, in your experience, it this a determinant topic on the succesfull of the reaction?
3. How do you handle the stringency of the reaction? I have had the both sides of this problem: reactions in wich no sample give signal (neither positive control), and reactions in wich all samples give signal (even negative controls). So, what do you think that is the best way to handle the stringency?
Thank you for your attention. I hope you can help me with your experience.