IP reactions were carried out in High stringency IP buffer supplemented with protease inhibitor cocktail, for 1 hr at 4°C.
Hybridisation was performed overnight at 65°C and the blot washed in high stringency according to instructions given by the manufacturer.
The filters were then washed for about 2 hrs at 55°C in high stringency buffer (0.1XSSC, 0.5% SDS, O.1% sodium pyrophosphate) with gentle shaking.
Slides were washed for 10 min each at 50°C in low stringency buffer (2× SSC; 0.5% SDS), at room temperature in medium stringency buffer (0.2× SSC; 0.5% SDS) and in high stringency buffer (0.1× SSC), then N2 dried.
Hybridizations were carried out in high stringency conditions at 68°C overnight using 50 mL of fresh hybridization buffer supplemented with a minimum of 10cpm purified and denatured probe per mL of buffer.
Slides were washed two times for five minutes each in low stringency wash at 42°C (2× SSC, 0.1% N-lauroysarcosine), followed by two washes for 5 min in medium stringency wash (0.1× SSC, 0.1% N-lauroysarcosine) at RT and 2 washes for 5 minutes each in high stringency wash solution (0.1× SSC).
Moreover, except for two faint additional signals with (AATAC 3 and (AAGAC 3, the results obtained here were in complete agreement with previous localizations involving clones containing the satellite repeats as probes in high stringency in situ hybridization experiments using other flies stocks ([ 10, 11] and Figures 1 and 4).
Following hybridization, membranes were washed twice in low stringency conditions (25°C, 5 min, 2× SSC buffer with 0.1% SDS) and twice in high stringency conditions (68°C, 15 min, 0.5× SSC with 0.1% SDS), where 1× SSC comprises 150 mM NaCl and 50 mM sodium acetate, pH 7.0.
Following hybridization, the slides were washed once in low stringency wash buffer (1× SSC, 0.2% SDS) preheated to 42°C for 5 minutes, once in high stringency wash buffer (0.1× SSC, and 0.2%SDS) for 5 minutes at room temperature, and then once in 0.1× SSC for 5 minutes at room temperature.
