Can the dot blot protocol be modified if I don't have a dot blotting apparatus for use with your 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692?

If a dot blotting apparatus is not available for use, we recommend denaturing your DNA in a lower volume/higher concentration (see modified steps below):
Section B. Dot Blot Modified Steps:
1. Dilute fragmented genomic DNA to 400 ng/ul in 100 ul of nuclease-free water. Then denature DNA by adding 100 ul of 2X DNA Denaturing Buffer and incubating at 95C for 10 min.
2. Add 200 ul of 20X SSC buffer and immediately chill on ice for 5 min. DNA concentration of the solution is 100 ng/ul.
3. Set up a series of six 2-fold dilutions by adding 200 ul of the DNA solution, starting with the DNA solution in Step 2, to 200 ul of nuclease-free water. This will generate seven DNA samples containing 200 ul DNA at concentrations of 100 ng/ul, 50 ng/ul, 25 ng/ul, 12.5 ng/ul, 6.25 ng/ul, 3.125 ng/ul and 1.563 ng/ul.
4. Spot 10 ul of each of the seven dilution samples onto the nylon membrane, leaving the last well for nuclease-free water only. The amount of DNA added to each well should then be 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, 15.625 ng and 0 ng respectively. Air dry the nylon membrane at room temperature until it is completely dry.
5. For UV-crosslinking of the membrane, the time depends on the model used for cross-linking. UV cross-link nylon membrane at 1200 J/m^2. This is the total amount of UV cross-linking to use. For most models, one can simply enter this value and the machine will cross-link accordingly.

Last updated: September 12, 2024