Southern Blot troubleshooting - Please help (Apr/13/2010 )

Dear all

I got in a few days an appraisal at work and I may be asked about troubleshooting for DNA Southern Blotting. I have found this picture but unfortunately I do not have the solutions for these problems. So, I have analysed the problem myself, explaining what I think it's the cause and what the potential solutions are. I would like people with more experience to give me a hand and tell me if I'm correct about the causes and solutions. Anyone else what to join me in this challenge? What would you do in each case? All the suggestions and ideas very welcome.

See the picture attached. These are the details of my troubleshooting.

A: Successful blotting: clean background and strong bands.

B: - Faint bands, nor marker visible: unsuccessful DNA-transfer to the membrane. Solution: Use new transfer solution and allow the transfer for a longer period. Check the gel following the DNA transfer to make sure that no DNA remains in the gel.
- Faint bands, nor marker visible: Pre-hybridisation or most likely washing for too long, most DNA has been washed. Solution: decrease the time of washing, decrease stringency, decrease temperature of washings...

C: -Spotty membrane: Blocking and/or hybridisation solutions were not evenly distributed. Solution: make sure that there is enough solution to cover the whole membrane and is rotating or shaking to get an even distribution. Make sure that parts of the membrane are not on the top of each other.

D: -Spotty membrane: membrane got dry after hybridisation. Solution: make sure that enough solution is available to cover the whole membrane and do not allow to get dry for long periods.
- Spotty membrane: powder from the gloves. Solution: Use powder-free gloves.

E: 1- Spots in the DNA lanes: bubbles during the DNA transfer. Solution: roll the membrane and Whatman paper to make sure there are not bubbles between the membrane and the gel
2- No clear bands, all DNA has been radioactively labelled. Solution: 2- Use a fresh 1- Pre-hybridase the membrane for longer to decrease unspecificity. 2- Use a freshly-made blocking solution (Church and Gilbert) 3- Decrease the amount of DNA.

Thanks a lot for your help.

Any kind soul want to give me a hand? Do you think that my hypotheses are correct?

Please, please and more please...!!!!!

Your answers look OK to me.

Thanks bob. It's good to have some reassurance. It's difficult to find practical exercise to practice my troubleshooting skills.