We have been troubleshooting the DIG-labeled southern blotting kit from Roche with no success. Here is a little background on what we have been able to achieve and what we have tried.
We are unable to detect a single copy section of the genome using this method. When we stain our membrane, our DIG labeled molecular weight ladder exposes nicely, and we will range from no staining to just a nondescript staining of the lane with DNA (no specificity, just background where DNA is present).
1. Probe design: we have tried two different methods of creating the probe. First, we tried PCR amplifying a segment (tried a 300bp and a 700bp) from the target genome. After PCR amplification, PCR cleanup was performed and probe was labeled using the klenow enzyme provided in the kit. The second method we used was to gel extract a homologous portion of DNA from a plasmid and label this using the labeling kit. Both methods were able to achieve adequate labeling based on the dilution series in the kit manual.
2. DNA digestion - we have been digesting overnight using a 4-cutter overnight. We have not been using spermidine - could this be an issue? We are cutting 10mg of dna and are loading all of this into a single lane.
3. Transfer of DNA - we are using the + charged nylon membrane from Roche. We have tried both a neutral transfer + UV cross-link and an alkaline transfer (no cross-link). When staining the gel post-transfer there is still some residual DNA on the gel but significantly less than before transfer.
4. As a control, we have run unlabeled probe on the gel to see if the probe would anneal to this, which it did very strongly.
Questions - at this point, I am not sure why there is background staining wherever DNA is present - is this normal, or does this represent an issue with the DNA labeling process? Do you think spermidine during the digest would make a significant difference? Do you think this merely represents not enough DNA on the nylon membrane itself, is there anything that we could do differently?
This has been a bit more challenging than expected, and I welcome any suggestions or thoughts from those of you out there with DIG Southern Blot experience.
Did you check your probe by dot-blot test as suggested in the manual to see if you have the required specific activity? The labeled probe is quite stable at -20, but I have had problems before with the anti-DIG antibody going off quite quickly. I prefer using the biotin-streptavidin method as the streptavidin-AP is more stable than the anti-DIG AP. Also, this method works fine using Church-Gilbert hybridisation buffer, a much cheaper and easier to prepare alternative to the DIG hyb solution.
- Badges
- Science topic