MICPRF@latvax8.lat.oz (07/05/90)
Greetings molbio gurus, Here is a problem that is puzzling us. We are working with Dictyostelium nuclear DNA for various purposes and noticed unexpected degradation of the DNA in our preps. occasionally. The problem has now been traced to heat. The DNA is ok at temperatures up to 52 degrees C, but at the next highest temperature we tested, 68 degrees C, a 30 minute or 1 hour incubation leads to complete degradation of the DNA. This is occurring in normal TE buffer, and since we do not work with thermophilic organisms of any kind, it seems unlikely to me that it is enzymatic. Everything we work with is, of course, sterilized appropriately. This has no effect on our ability to probe genomic Dictyostelium DNA with bacterially grown probes. Here the genomic DNA is already cut, denatured and bound to the nitrocellulose before hybridization at 68-70 degrees C. The DNA is from a nuclear prep. after gentle Triton lysis of the cells and has been phenol extracted several times, then chloroform extracted and ethanol precipitated. While it doesn't affect Southern blot analysis, there are obviously other situations where it becomes a nuisance to say the least. Does anyone out there in Netland know what the cause might be, and how it can be avoided? Regards, Paul Fisher (micprf@latvax8.lat.oz.au)