Each of the three regions was detected in all VLM-producing strains under conditions of high stringency (Fig. 1c).
Replica plating was performed under conditions of increasing stringency according to the manufacturer's suggestions and subsequently analysed for growth on –Leu/-Trp/-His, -Leu/-Trp/-His/-Ade synthetic drop out (SD) media.
After a succession of one hour washes (2xSSC/1 mM DTT, 50°C; 0.2xSSC/1 mMDTT, 55°C; 0.2xSSC/1 mM DTT, 60°C), the tissue was treated with RNase-A and washed under conditions of increasing stringency, including a 30 min wash at 60°C in 0.1xSSC.
Clearly, validation of results will be even more critical in cross-species microarray experiments because of questions surrounding cross-hybridisation of related genes under conditions of reduced stringency.
Thus, we tried to estimate the approximate size of the goat and sheep V1R gene repertoire by genomic Southern blot analysis under conditions of low stringency.
The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency.
The digested genomic DNAs were size fractionated by agarose gel electrophoresis, blotted on nylon membrane (Biodyne Plus, Pall, MI, USA), and hybridized under conditions of low stringency.
Probes were hybridized overnight under conditions of high stringency at 65°C and detected by using chemiluminescence as recommended by the manufacturer.
Unlike conventional PCR-based analyses, this approach uses oligonucleotide primers of an arbitrary sequence to start DNA strand synthesis under conditions of low stringency.
Under conditions of reduced stringency, certain lanes exhibited more than the two expected bands (data not shown), indicating the existence of related sequences within the genome.
