, Defrost 1µL of biotinylated target stock oligonucleotides (antisense nucleotides)

, Dilute the target oligonucleotides at 10fmoles/µL in TE 1X, p.8

, Add 1.5 µL of each coupled microbeads stock to 998,5 µL of TMAC 1.5X hybridization Solution)

, Mix the « Microbeads working Mix » by vortexing 20 s and sonication during 20 s

, In each assayed well of a 96-well plate, including the negative control add 33µL of « Microbeads working Mix

, in the negative control well(s), add 17µL of TE 1X, p.8

, In each tested well, add the mixture of biotinylated complementary oligonucleotides (5 to 200 fmoles) and TE 1X, pH=8 up to a total, vol.17

, Cover the 96-well plate to prevent evaporation and incubate at 95-100°C during 3 min to break all oligonucleotides secondary structures

, Incubate the 96-well plate at the same temperature as the one used for the PCR-product hybridization assay (52°C for TB-SPOL and 59°C for STM-CRISPOL) during 20 min (cf. section 4, vol.8

, Centrifuge the plate during 1 min, eliminate as much as possible of the supernatant (25-35µl) by pipeting carefully, replace with 25-35 µl of TMAC 1X

, Reporter Mix » by adding 4µL of Streptavidin-R-phycoerythrin in 996µL of TMAC 1X hybridization solution to obtain a 4µg/ml solution (4/1000 of 1mg/ml

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