1.The Oligo probe was dissolved in a 96-well plate with 0.5×TE pH8.0 to a concentration of 200um. 2.Transfer 7.5ul of each probe to a new 96-well plate, incubate at 37°C with 1000units of T4 PNK phosphorylase in 1×T4 PNK phosphorylation buffer solution for 2 hours, and the total volume is 15ul. 3. Transfer the phosphorylated probe to 30°C, add 4.5ul of 500μM linker sequence, use 1000units of T4 DNA ligase and incubate for 20 hours in 1×T4 DNA ligation buffer, the total volume is 35ul. 4. After the probe is ligated, precipitate with isopropanol and dissolve in 50ul of 0.1M NaOH solution. 5. Modified with 8mM alkylating agent for 2 hours at 65°C, then precipitated and dissolved in 0.1M NaOH (containing 0.5M NaCl) at a concentration of 350-400ng/ul. 6. Transfer the probes to a Genetix 394-well plate and place them in the sample wells of the Qarray2 chip spotter. 7. Start the Qarray2 spotting machine to automatically spot samples. After completion, the chip is subjected to constant temperature and humidity treatment to help the probe be better fixed, and then the residual salt on the surface is washed off for use. The remaining probes were sealed with aluminum foil and stored frozen at -20°C.
Less...
More...