Was wetting done with MeOH or Distilled water?

The volume of dispensing can be an issue

@ Azad Ismail Saheb wetting was done with 6x SSC. I added 100uL of DNA to each well

I always use nitrocellulose for dot blot, although I do RNA dot blots but it's the same protocol so doesn't matter. I always wet the filter and membrane in 1X SSC for 15 min before applying the sample and never get the "leaking". Also, make sure your apparatus holds the membrane and filter assembly tightly, if not you can increase the number of filter papers under the membrane to make it tight enough. Hope it works for you.

Great thank you so much @ Mansi Garg I shall try this. Could you please share the protocol with me if that's okay? Also do you UV crosslink or bake the membrane at 80°C?

Hello

I haven't done Dot blots with DNA, but in our lab we have observed some dificulties using BioRad apparatus. Despite perfect circles and low background signals if vacuum is not perfectly sealed the sample can be unevenly absorbed and appears like leaky blots with halo or moon appearance. Recently we've prefered perform Dot blots in a 24 well-plate format, you just need to cut pvdf circles, activate the membrane, add your protein/DNA (50ng is suficient but you can try 50 to 100ng) and allow it to absorb overnight at 4°C, then you just continue with blocking and antibody incubations. With this format we've used sample volumes from 5 to 100ul (we recomend 30ul), and when working with volumes >10ul you just need to add these from 10 to 10ul and remove the excess of volume that is filtered through the membrane.

With conditions like these we've observed positive results with as little as 50ng recombinant viral protein & 300ng of polyclonal antibody (150ng is enought but as we use these in a next step for cell culture assays and we have to avoid NaN3, antibody titer drops fast and appears like poor signal & higher background) or 5x10e4 UFPs and just 30ng of monoclonal antibody. The complete 24 well-plate can be revealed in a BioRad's Chemidoc System and for primary and secondary antibody incubations you just need to use 300ul, so you can save antibody and test diferent amounts of antibody or protein/DNA.

Here you have the reference inspired us to adapt the format to our lab:

Dot-immunobinding assay - PubMed (nih.gov)

Hope it helps

coudn't paste the link, here's the PMID

pubmed.ncbi.nlm.nih.gov/19378048/