- Quickly decant the wells and wash with 300μL of wash buffer
for three times, with quick decanting in between. - Centrifuge the plate upside-down for 1 min at 600gafter the
final wash and decant. - Add 100μLof1μM adaptor probe in hybridization buffer to
the wells. - Seal the wells with tinfoil sealing film and incubate at 46C for
1h. - Tear the film, then quickly decant the wells and wash with
300 μL of wash buffer for three times, with quick decanting
in between. - After the final wash, centrifuge the plate upside-down for 1 min
at 600gto remove residual liquid. - Mix 120μL of hybridization buffer, 15μL of probe X1*, and
15 μL of probe X2 (the final concentration of each hairpin
probe is 1μM). - Add 100μL of the above mixture to the capture plate and seal
the wells, incubate at room temperature for 2 h. - Repeat wash steps (steps 9and 10 ).
- Mix 120μL of hybridization buffer, 15μL of probe A1, and
15 μL of probe A2. - Add 100μL of above mixture to the capture plate, seal and
incubate at room temperature for 2 h. - Repeat wash steps (steps 9and 10 ).
- Add 150μLof4SYBR Green I in 4SSC buffer to the
reaction wells, and then incubate at room temperature for
15 min in the dark place. - Quantify the resulting fluorescence with a plate reader.
4 Notes
- Ethidium bromide should be kept away from light in room
temperature. As EtBr is a known mutagen, be careful when
operate with this chemical and during the process of agarose gel
electrophoresis. - Take the plate out from refrigerator and warm to room tem-
perature before use. The capture plate is sealed with plastic film,
so tear the film of specific wells where you want to add your
sample to. - Blood sample and Proteinase K should be thawed at 4C and
operated on ice throughout the procedure.
194 Yao Xu and Zhi Zheng