Southern blot help--no signal - for bacterial KO verification (May/17/2010 )

Hi everyone,

I have been struggling with failed southern blots... Any advice is appreciated!

I need to check if my gene is deleted in my bacterial knock-out strain, I used the enzyme EarI, which can double-digest the genomic DNA region: it produces a 2kb DNA fragment for the WT strain, and a 3kb DNA fragment for the KO strain. I made sure the DNA (I use plenty of DNA) was successfully transferred to the membrane, and also that my probe (I use plenty of probes too) was successfully labelled because I knew it reacted strongly with the detection reagent.

I found some on-line protocol says that the depurination step will fragment any >3kb DNA pieces into smaller fragments and help with transfer efficiency. I wonder if this causes my problem since I have to detect a 3kb fragment. Shall I stop using the depurination solution and transfer my gel for a longer period of time?

Thank you in advance for your advices.

ahhdust

You should simplify your debugging by starting with dot blots rather than southern blots. Take your genomic dna and do serial dilutions, spot on a membrane, and detect to determine if your hybridization conditions are correct. You could also check the ability to detect after depurination. Only when this is working do you worry about gels, cutting, and transfer efficiency.

phage434 on May 17 2010, 10:16 AM said: