DNA Array

DNA matrix products

I. Materials required
Buffers
- Prehybridization solution: 5xSSC, 0.1%SDS, 0.1%BSA
- 2x hybridization solution: 40-70%Formamide, 10xSSC, 0.2%SDS, 0.02%salmon sperm DNA (optional)
- Wash solution 1: 2xSSC, 0.1%SDS
- Wash solution 2: 0.1xSSC, 0.1%SDS
- Wash solution 3: 0.1xSSC
Material

- Boxes for prehybridization and hybridization
- Hybridization chamber
- 20 µl to 1,000 µl single-channel micropipettes with disposable tips
- 5 ml and 10 ml graduated pipettes
- Water bath
II. Duration of experiment
- 30 min pre-hybridization
- 12 to 16 hours hybridization
- 10 min washes
- TOTAL: 13 to 17 hours
III. Procedure
Pre-hybridization
1. Prepare an appropriate volume of Prehybridization Solution.
Note: the volume required depends on the box used; the level must reach the barcode on the slide.
2. Pre-heat solution to 55°C.
3. Place slides in solution and incubate for 30 min with agitation.
4. Rinse 5 times with H2O.
Standard hybridization
1. Prepare 2x Hybridization Solution
2. Mix labelled sample with 2x hybridization solution
3. Adjust volume to 1x concentration and mix.
4. Incubate 2 min at 95°C
5. Place on slide in hybridization chamber
6. Incubate 12 to 16 hours at 42°C
Washing Hybridization
1. Prepare wash solutions 1, 2 and 3.
2. Remove slide from hybridization chamber and place in wash solution 1.
3. Incubate under agitation for 5 min.
4. Transfer to solution 2 and incubate with shaking for 5 min
5. Transfer to first volume of solution 3 and incubate with shaking for 1 min
6. Repeat step 5.