9-mer Hybridization Protocol
Visualization of printed DNA as a quality control check can be easily performed
by hybridizing the array with a Cy-labeled random 9-mer sequence at room temperature.
Basic Protocol:
- use 150 pmol of Cy labeled 9-mer in a regular Hybridization mix
- hyb at Room Temperature 3-5 min.
- wash slide
- scan
for 20 ul 9-mer Hyb Mix:
| Target | Amount | Stock |
| 4x SSC | 4 ul | 20x |
| 1 mg/ml poly-dA | 2 ul | 10 mg/ml |
50 mM HEPES pH 7
(or Tris pH 7.5) | 1 ul | 1 M |
| 0.2% SDS | 0.4 ul | 10% SDS |
| 7.5 uM Cy3 random 9-mer | 150 pmols | ? |
| Total Volume to 20 ul with H2O |
- Briefly heat probe to 90 deg C.
- Cool by Spinning in Microfuge (don't place on ice or SDS will precipitate)
- Pipet probe onto slide and use coverslip as you would for a normal hybridization.
- Allow to incubate at room temperature for 3 to 5 minutes.
- Wash slide in 2X SSC 0.2% SDS
- Wash slide in 0.05X SSC
- Dry slide by spinning slide rack or by placing slide in 50 ml Falcon tube, and spinning at 500-1000 RPM for 5 min.
Notes:
The Tm for a 9-mer ranges from about 4 to 40 degrees Celsius. The average will likely be below room temperature. So the things to consider with this protocol are Temperature, salt concentration, and oligo concentration.
Chris Seidel Sept. 1999
