Amersham protocol for processing CodeLink (modified oligo) slides:
Overview
After printing amino-modified long oligonucleotides, Amersham CodeLink slides are processed to bind the probe DNA to the slide and prevent non-specific hybridisation to the substrate. The outlined protocol is based on the method recommended by Amersham (http://www1.amershambiosciences.com/).
Equipment and material
- Amersham CodeLink slides (Amersham; Cat. No. 300011)
- Slide staining rack (Philip Harris; Cat. No. B52651)
- Slide staining trough (Philip Harris; Cat. No. B52649)
- 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)
- Blocking Solution: 50 mM ethanolamine, 0.1 M Tris, (pH 9.0), 0.1% SDS
- Wash solution: 4 x SSC, 0.1% SDS
- Ultra pure water (do not use MilliQ water)
- Standard photographic air duster
- Air tight plastic box (30 x 30 x 18 cm) with lid
Protocol
For best results, perform steps 4 onwards just before hybridisation.
- Incubate slides in a chamber with 65 to 75 % relative humidity overnight:
- Within an air tight plastic box add 100 g solid sodium chloride to 50 ml water
- Allow slides to dry at room temperature for 30 minutes
- Slides that are not needed for one 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 50 °C in a waterbath
- Prepare and preheat the wash solution to 50 °C in a waterbath
- Pour the blocking solution into a slide staining trough and then transfer the slides to a slide staining rack and place this rack in the staining trough
- Place the trough containing the slides on the orbital shaker at 50 rpm for between 20 to 30 minutes at room temperature
- Remove rack, blot off excess solution by placing on a piece of tissue.
- Transfer the slides to a staining trough containing RO water, then move up-and-down 30 times (to rinse the slides)
- Repeat step 9
- Pour the wash solution into a slide staining trough, then transfer the slides to this staining trough
- Place the trough containing the slides on the orbital shaker at 50 rpm for between 20 to 60 minutes at room temperature
- Remove rack, blot off excess solution by placing on a piece of tissue.
- Transfer the staining rack to 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 5 minutes
- 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 (04-07-2005).
