The RNA was blotted with 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) onto a positively charged nylon membrane (Boehringer GmbH, Mannheim, Germany).
Hybridization to 5′ (P -phosphorylated oligonucleotides was carried out over night at 62°C, followed by consecutive washing steP -phosphorylatedSSC, 1% SDS/1 × SSC and 0.1% SDS/1 × SSC.
After hybridization, larvae were washed several times in 4 × Wash (25%% formamide, 4 × SSC, 0.1 % Tween 20), 2 × Wash (with 2 × SSC) and 1 × Wash (with 1 × SSC).
Blots were washed 2 3 times with 2 × SSC, 0.2% SDS and once with 1 × SSC, 0.1% SDS.
After hybridisation, membranes were sequentially washed at room temperature (RT) twice for 30 min in 2 × SSC, 0.1% SDS, followed by two washes in 1 × SSC, 0.1% SDS.
The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer.
The optimal annealing temperature for nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify the gene was 61 °C and optimal hybridization occurred when buffer 1× SSC and 0.5% SDS were used at 50 °C.
After hybridization, slides were washed twice in 1× SSC, 50% formamide at room temperature, four times in 2× SSC, 50% formamide at 42°C, and rinsed in 1× SSC.
The arrays were washed at room temperature in 5 successive buffers, i.e., 1× SSC+0.2% SDS, 0.1× SSC+0.2% SDS, 1× SSC, 0.1× SSC and 0.001× SSC, each step lasting 4 minutes.
The arrays were then washed sequentially in 400 ml of 2× SSC with 0.1% SDS, 1× SSC, and 0.2× SSC.
