Tmp chromogenic solution for elisa

Single-target Beads-ELISA detection reaction (20 µL) contained 1× Binding buffer, 15 nM sgRNAa, 15 nM sgRNAb, 30 nM dCas9 protein, 1-4×10 4 Beads@oligo, 0.5 µM oligo re-biotin (Table S3), 40 U RNA Enzyme inhibitor, and DNA sample (see figures for amount). Multi-target Beads-ELISA detection reaction (20 µL) contained 1× Binding buffer, 15 nM each of sgRNAa, 15 nM each of sgRNAb, 15× N nM dCas9 protein, 1-4×10 4 Beads@oligo, 0.5 µM oligo re-biotin, 40 U RNase inhibitor, and various amount of DNA sample (see figures); where N is the number of targets detected. The reaction was incubated at RT for 15 min in rotation. The beads were then washed three times with a washing buffer (1× dCas9 buffer contained 10 U of RNase inhibitor and 0.5% BSA), in which 1× dCas9 buffer can be purchased from New England Biolabs as NEBuffer 3.1 or prepared at home. The beads were incubated with 20 µl washing buffer containing of 8 ng of HRP-conjugated Streptavidin (Sangon Biotech, Shanghai) for 3 min. The beads were washed three times with the washing buffer and finally re-suspended in 30 µl of washing buffer. The beads were transferred into microplate and added 50 µl of TMP chromogenic solution for ELISA (P0209-100ml; Beyotime). The beads were incubated at RT for 10 min. The microplate was read at 630 nm and imaged with a BioRad gel imager in staining-free mode.

An amino-modified oligo RE-NH 2 (Table S3) was covalently coupled to the DNA-BIND 96-well microplate (Corning) according to the instructions of manufacturer to prepare the oligo-coated microplate.
Single-target Microplate-ELISA detection reaction (80 µL) contained 1×Binding buffer, 15 nM sgRNAa, 15 nM sgRNAb, 30 nM dCas9 protein, 0.5 µM oligo re-biotin (Table S3), 40 U RNA Enzyme inhibitor, and various amount of DNA sample (see figures). Multi-target Microplate-ELISA detection reaction (80 µL) contained 1× Binding buffer, 15 nM each of sgRNAa, 15 nM each of sgRNAb, 15 nM × N dCas9 protein, 0.5 µM oligo re-biotin (Table S3), 40 U RNase inhibitor, and various amount of DNA sample (see figures); where N is the number of targets detected. The reaction was added to the oligo-coated microplate.
The microplate was incubated at RT for 15 min on a horizontal mixer and then washed three times with the washing buffer. The microplate was added with 100 µL of washing buffer containing 8 ng of HRP-conjugated Streptavidin and incubated at RT for 3 min. The microplate was then washed three times with the washing buffer. The microplate was then added with 30 μL of washing buffer and 50 μL TMP chromogenic solution for ELISA (Beyotime). The microplate was incubated for 10 min. The microplate was read at 630 nm and imaged with a BioRad gel imager in staining-free mode.