Post Processing of DNA microarray slides made with PCR products and long oligos (70mers)
This protocol is for microarrays containing two types of targets on the same slide: PCR products and long oligos (70-mers). It combines aspects of the protocols for each type of target. Slides are UV crosslinked, and the shampoo and final rinse are both done at 50° C.
Set up three baths for slides:
- 400 ml Shampoo Soln, 50 °C
- 400 ml H2O
- 400 ml 95% Ethanol
|Preheat Shampoo solution to 50 °C: ||Shampoo Soln 400 ml|
|60 ml 20x SSC|
|4 ml 20% SDS|
|336 ml distilled H2O|
Succinic Anhydride Blocking
- Mark slides on the back with diamond scribe pen.
- Expose slides to 65 mJ of UV irradiation.
- Dunk slides into Shampoo Solution at 50 °C, plunge for 15 seconds, incubate for 5 min.
- Transfer to 400 ml H2O for a few seconds to rinse
- Transfer to 400 ml 95% EtOH for a few seconds for drying
- Spin slides at 500 RPM, 5 min., Room Temperature!
|Materials: ||335 ml 1-methyl-2-pyrrolidinone|
|5.5 g Succinic Anhydride|
|15 ml 1M Na Borate pH 8 (premeasure into 15 ml conical)|
| || |
| Rotating platform shaker|
| Empty slide dish|
| 400 ml 95% EtOH bath|
| Room Temperature H2O bath in beaker large enough to rotate slide rack|
Notes: Single distilled water is fine for the H2O rinse steps. For the second rinse coming from pyrrolidinone to water, a 4 liter beaker works well for rotating the slide rack back and forth.
- Place 335 ml 1-methyl-2-pyrrolidinone into 500 ml beaker with stir bar
- Add 5.5 g Succinic anhydride
- When the last crystal dissolves add 15 ml 1M Na Borate pH 8.0 and transfer to a slide dish
- Plunge slide rack rapidly into blocking solution, plunge mix up and down for 30 seconds
- Shake gently on rotator for 15 minutes
- Drain blocking solution off slides and transfer to 50 °C water bath
- Incubate 60 seconds
- Transfer to 95% Ethanol bath to rinse
- Spin slides dry in the centrifuge, 500 RPM, 5 minutes, Room Temperature
- Store the slides in a dust free plastic slide box, they are ready for immediate use.