In this report, we present refinements to a previously published technique for slot blotting, a method we have used for detecting and measuring quantitative polymorphisms in maize DNA sequences. We have found this technique to be useful for studying copy number variation of repeated sequences among various maize varieties (Rivin et al., Genetics 113:1009, 1986) and for examining very low copy numbers of Robertson's Mutator transposon-homologous sequences in non-Mutator maize lines (Chandler et al., Genetics 114:1007, 1986). A detailed protocol for the technique and data analysis has been published (Rivin, Meth. Enz. 188:75-86, 1986).
We have modified our procedure by adapting the method of Reed and Mann (NAR 13:7207, 1985) for Southern blotting onto positively charged nylon membranes. There are two major differences in the modified method: denatured DNA samples are not neutralized prior to loading onto the filters and baking filters in vacuo prior to hybridization is eliminated. These changes represent a great savings in time and ease of handling and the resulting blots give very sharp signals that are stronger than with the original method.
Modified slot blot method:
1. Preparation of the apparatus: The slot blot apparatus is soaked in a 0.4 M NAOH solution prior to use. A positively charged nylon filter (we use Genatran) is wetted in distilled water and then briefly rinsed in 0.4 M NAOH. The apparatus is assembled as described previously.
2. Preparation of DNA samples: The DNA samples to be loaded are denatured by adding NAOH to a final concentration of 0.2 M and heated at 95C for 2 minutes.
3. Loading samples: The denatured samples are cooled on ice and vortexed before loading into the slots. After the entire sample has blotted, the well is rinsed through with 200 microliters of 0.4 M NAOH.
4. Handling the filter: After the apparatus is disassembled, the membrane is soaked for 5 minutes in 5 X SSC. It is then ready for prehybridization. The filters do not need to be baked. They can be stored after air drying.
Donna Hazelwood and Carol Rivin
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