Southern blot help--no signal
Posted 17 May 2010 - 07:10 AM
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.
Posted 17 May 2010 - 09:16 AM
Posted 18 May 2010 - 05:45 AM
Thank you! I wonder if the probe can bind to the diluted genomic DNA without denaturing?