Full Moon Biosystems (FMB) protocol for processing FMB cDNA slides:
Overview
After printing non-modified PCR amplified gene-specific cDNA clones the Full Moon Biosystems (FMB) cDNA slides are then processed to bind the denatured probe DNA to the slide and prevent non-specific hybridisation to the substrate. The outlined protocol is based on the method recommended by Full Moon Biosystems (http://www.fullmoonbio.com/).
Equipment and material
- Full Moon Biosystems cDNA slides (Full Moon Biosystems; Cat. No. AS 50)
- Slide staining rack (Philip Harris; Cat. No. B52651)
- Slide staining trough (Philip Harris; Cat. No. B52649)
- UV crosslinker (Ultraviolet products; CL-1000)
- Orbital shaker (Stuart Scientific; mini orbital shaker SO5)
- 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)
- Bovine serum albumin (BSA) fraction V (Sigma; Cat. No. A-7906).
- Sodium dodecyl sulfate (SDS), molecular biology grade (Sigma; Cat. No. L-4390)
- Blocking Solution: 4xSSC, 0.1%SDS, 1%BSA
- MilliQ water
- Standard photographic air duster
Protocol
For best results, perform steps 4 onwards just before hybridisation.
- After arraying, UV cross-link the slides with the cross linker set at 4000 (x100µJ) = 400mJ
- Allow slides to dry at room temperature for 30 minutes
- Slides that are not needed for one month or two months can be stored at this stage:
- Place the slides in a clean microscope slide box
- Then place the microscope slide box in a pastic bag and seal this bag
- Store the sealed bag at 2 to 8 °C for 3 to 6 months
- Meanwhile prepare and preheat the blocking solution to 55 °C in a water bath
- Pour the blocking solution into a slide staining trough
- Transfer the slides to a slide staining rack and place the rack into the slide staining trough
- Place box on shaker at 50 rpm for 20 minutes at room temperature
- Remove rack, blot off excess solution by placing on a piece of tissue.
- Place the slides in the staining rack in the plastic box filled with 2.5 L ultra pure water and put the lid on
- Place the plastic box with the slides on the orbital shaker at 50 rpm for 15 minutes
- Remove rack, blot off excess solution by placing on a piece of tissue.
- Repeat steps 8 to 10 twice (three water washes in total)
- Transfer slides from the rack to a microscope slide box with fresh tissue in the base
- Centrifuge at 650 rpm for 15 minutes in a microtitre centrifuge to dry the slides
- Remove any water droplets from the slide using an air duster
- Store in a clean sealed slide box at room temperature and in the dark (one to two months) until ready to hybridise
R. Auburn (05-06-2006).
