Eccles:random panomer hyb

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Random Panomer Hyb

Useful to check integrity of spots on spotted arrays. Original protocol from Chris Seidel when at Berkeley, and now used by Molecular Probes for their Alexa Fluor-labelled random oligos. The following is straight from the Molecular Probes protocol. We have Panomer 9 random oligodeoxynucleotide Alexa Fluor 555 (P21687): use as instructed below, and see Aaron for it's whereabouts.

Hybridisation to Microarrays

  1. Make a 100 µM stock solution by adding 100 µL of deionized water (dH2O) or TE buffer (10 mM Tris, 1 mM EDTA, pH 8) to the vial containing the dried Panomer 9 oligonucleotide.
  2. Prepare the Panomer 9 hybridization solution by diluting the stock solution of the Panomer 9 random oligodeoxynucleotide 13.3-fold into a standard hybridization buffer for a final concentration of 7.5 µM. 50 uL for 50x22 mm lifterslips, 70 uL for 60x25 lifterslips.
  3. Briefly heat the Panomer 9 hybridization solution (prepared in step 2) to 90°C for 15-­30 seconds, then cool it gently by centrifuging the tube in a microcentrifuge (do not place the solution on ice, because the SDS will precipitate).
  4. Pipet the Panomer 9 hybridization solution onto the microarray slide, under a lifterslip or coverslip.
  5. Allow the hybridization to proceed at room temperature for 5-­10 minutes.
  6. Remove the coverslip, and wash the microarray in a solution of 2X SSC, 0.2% SDS for 40 seconds.
  7. Immediately wash the microarray in 0.05X SSC for 20 seconds.
  8. Dry the microarray by centrifugation -- place it in a slide rack or a 50 mL centrifuge tube, and centrifuge at low speed (500-­1000 rpm) for 3-­5 minutes.
  9. Read the fluorescence signal using the appropriate excitation and emission wavelengths e.g. 532 on the Axon 400B scanner.

Removal of Bound Panomer 9 Oligos from Microarrays

  1. Place the microarray slide in an excess of dH2O and agitate gently for one minute. If some fluorescence still remains, lengthen the wash time or increase the stringency of the wash by raising the temperature.