Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays

1. Adjust the volume to 20 μl with TE (see Tip #3).
2. Add the following for the final probe preparation (see Tip #4):  4.25 μl of 20X SSC,  0.75 μl of 10% SDS (see Tip #5)
3.  Denature the probe by heating for 2 min at 100°C.
4.  Incubate at room temperature for 15 to 20 min.
5.  Centrifuge at 14 krpm for 10 min.
6.  Place 24 μl of the probe on the array underneath a 22 mm x 40 mm glass cover slip (see Tip #6).
7.  Prepare a slide chamber with the humidity maintained by a small reservoir of 3X SSC by spotting approximately 3 to 6 μl of 3X SSC at each corner of the slide (as far away from the array as possible).
8.  Hybridize at 65°c for 14 to 18 hr in the slide chamber.
9. Set up the 200 ml of washes in 250 ml chambers as follows: Wash I: 2X SSC with 0.1% SDS, Wash II: 1X SSC (prepare two chambers),  Wash III: 0.2X SSC (see Tip #7)
10. Blot-dry the exterior of the chamber with towels and aspirate any remaining liquid from the water bath. Aspirate the space between the two chamber halves.
11. Unscrew the chamber and aspirate within the holes to remove the last traces of water-bath liquid.
12. Place the arrays, singly, in a rack, inside the Wash I chamber (a maximum of four arrays at a time).
13. Allow the cover slip to fall, or carefully use forceps to aid the removal of the cover slip if it remains stuck to the array. DO NOT AGITATE until the cover slip is safely removed. Then agitate for approximately 15 sec.
14. Remove the array with the forceps. Rinse in a Wash II chamber (without a rack).
15. Transfer to a Wash II chamber containing a rack (see Tip #8).
16. Wash the arrays by submersion and agitation for 30 sec in the Wash II chamber, then transfer the entire slide rack to the Wash III chamber.
17. "Spin-dry" by centrifugation in a slide rack in a Beckman GS-6 tabletop centrifuge at 600 rpm for 2 min.
18. Scan the arrays immediately.

Microarray Probe Preparation Materials Needed:

• SDS
• EDTA
• Sodium Citrate
• tRNA
• dCTP
• dTTP
• dGTP
• dATP
• Magnesium Chloride
• RNA, PolyA
• DNA, Cot1 human
• Superscript II
• oligo dT, anchored
• Tris-HCl
• Oligo-dT
• DTT
• Tris
• Potassium Chloride
• Sodium Chloride
• Cy5
• Cy3