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.
for 20 ul 9-mer Hyb Mix:
- use 150 pmol of Cy labeled 9-mer in a regular Hybridization mix
- hyb at Room Temperature 3-5 min.
- wash slide
|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.
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