Quality control of printed microarrays by staining with SYBR 555
Overview
After printing, a random sample of microarrays is stained and then scanned so that we can asses the print quality and constancy. These checks include substrate defects, sub-grid and meta-grid positioning on the substrate, checking that all spots have been printed and spot morphology. Print batches that fail these quality control test are either used for teaching, or internal development.
Equipment and Reagents
- Between 2 and 8 microarrays from the printing batch to be tested
- 96% ethanol
- Slide staining rack (Philip Harris; Cat. No. B52651)
- Slide staining trough (Philip Harris; Cat. No. B52649)
- Hettich Rotina 35 microtitre plate centrifuge
- Microscope slide box (Merck EuroLab; Cat. No. 406/0286/00)
- Horizontal laminar flow work station (Jencons; Cat. No. 566-031)
- Paragon DNA microarray QC stain kit with SYBR 555 stain (Molecular Probes; Cat. No. P32930)
- Standard photographic air duster
Procedure (4 slides, per batch)
Prepare stain and wash buffers
- Add 95 ml 96% ethanol to stain buffer component B
- Add 190 ml 96% ethanol to wash buffer component C
Staining the slides
- Add 27 ml stain buffer to stain tube (provided) and add 5 µl SYBR_555, invert tube 5 times to mix
- Place up to 4 slides in the staining tube, incubate for 5 minutes at room temperature in the dark
- Remove slides from the stain and blot off excess solution
- Place slides into a fresh staining tube containing 27 ml of wash buffer, invert once to wash and then remove slide
- Place slides into a second staining tube containing 27 ml of fresh wash buffer and wash the slides for 5 minutes in an orbital shaker at 50 rpm in the dark
- Remove slides from the stain and blot off excess solution, transfer to a microscope slide box with tissue in the base
- Centrifuge at 1000 rpm for 5 minutes
- Remove any water droplets from the slide using an air duster
Scanning the slides
- Scan using the cy3-channel of a CCD or dual laser scanner
R. Auburn (17-02-2006).
