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Malaysia University |
POST-PROCESSING OF DNA MICROARRAYS PRINTED WITH PCR PRODUCTS
Post-Processing of DNA Microarrays Printed with PCR Products
Contributor:
Chris Seidel
Overview
The protocol describes the post-processing of DNA microarray slides that have been printed with PCR products. Post-processing of the recently printed DNA array ensures that the DNA is securely attached to the glass slide. This is analogous to attaching DNA to a nitrocellulose filter when performing a Western Analysis. Once properly attached to the glass slide, the DNA array will not wash away during the hybridization procedure (see Protocol ID#2217). The following method is similar to but not identical to a protocol for the preparation of cDNA arrays from the laboratory of Pat Brown at Stanford University.
Procedure
A. Rehydrating and Snap Drying of Microarray Slides (see Hint #1)
1. Select one full rack of array slides to be used for post-processing (usually 20 to 30 slides).
2. Ensure that the slides are identically oriented and are properly labeled (see Hint #2).
3. Make an etch with a diamond pen on the back of the slide to denote a guide for the cover slip (see Hint #3)
4. Rehydrate the slides by placing them face down over a humid chamber containing 3X SSC that has been warmed above room temperature (37°C).
5. Incubate the slides in the humid chamber for 1 to 5 min (see Hint #4).
6. Once the surface has been hydrated, the slides should be immediately placed array side up on a hot plate set between 100 to 140°C (see Hint #5).
7. OPTIONAL: UV Crosslink the DNA to the slides with 65 mJ of energy.
8. Return the slides to the slide rack.
B. Succinic Anhydride Blocking`
1. Measure out a 15 ml aliquot of 1 M Na Borate.
2. Combine 335 ml of 1-Methyl-2-Pyrrolidinone (Aldrich) and 5.5 g of Succinic Anhydride (Aldrich) in a dry, clean pyrex dish with a stir bar (see Hint #6).
3. Stir the solution to dissolve the Succinic Anhydride. As soon as the last flake of Succinic Anhydride dissolves, mix in the 15 ml aliquot of Na Borate.
4. Immediately plunge the slide-rack up and down into the solution quickly and evenly for 30 sec (see Hint #7).
5. Incubate the slides in the solution with gentle agitation on an orbital shaker for 15 min.
6. Remove the slide-rack and plunge the slides up and down in a slide dish containing 95°C ddH2O at room temperature.
7. Incubate in the ddH2O solution for 1 min.
8. Transfer the slides to a dish containing 95% Ethanol and plunge the slides in and out of the solution several times.
9. Dry the slides by centrifuging them in a table-top centrifuge at approximately 100 X g for 5 min.
10. Store the slides at room temperature in a dust free box until needed.
Solutions
1 M NaBorate
1 M Boric Acid (use solid Boric Acid, Fisher)
Adjust the pH to 8.0 using 5 M NaOH 
95% (v/v) Ethanol
SSC (3X)
0.9 M NaCl
pH 7.2
0.09 M Sodium Citrate
5 M NaOH
BioReagents and Chemicals
Ethanol
Sodium Chloride
Sodium Citrate
Sodium Hydroxide
Boric Acid
Succinic Anhydride
1-Methyl-2-Pyrrolidinone
Protocol Hints
1. The purpose of the rehydration and snap-dry step is to redistribute the DNA uniformly throughout the spots and to maintain that pattern. Slides are processed one at a time or a few in parallel with a few seconds between each manipulation. Perform these steps in an efficient manner and avoid awkward transitions. A slide can be grabbed from the humid chamber and placed on the heat block in one smooth, quick step. Notice how your hand can rotate with your forearm; keep this in mind while setting up the chamber and the heat block, placing things in ways that take advantage of how your body moves.
2. Label each slide by etching a number in the corner of theslide with a diamond pen, or use laminated labels. A low-cost label maker with laminated labels can be used for slide labeling. The labels will survive all aspects of slide processing. A number of labelers can print a series of labels with spaces so that slides can be easily labeled while sitting on the DNA arrayer.
3. Since the arrayed spots will disappear upon processing, a couple of etch marks on the back of the slide can guide the placement of the cover slip. This is easily accomplished by placing two plastic pipette tip boxes close together as an etch support, and placing the slide upside down across the two boxes such that the area of the spots is facing down between the boxes and is thus untouched.
4. The surface of the slide will fog up with condensation as the array spots become rehydrated (the spots will glisten). Do NOT rehydrate to such an extent that the spots begin to run together. Placing a desk lamp over the humid chamber will heat the liquid and foster condensation on the cooler glass surface.
5. At the right angle, one can observe the condensation rapidly disappearing from the slide surface; this is referred to as snap drying. It usually takes less than 5 seconds.
6. Confirm that the level of the liquid is deep enough to submerge a slide rack.
7. Move rapidly when submerging the slides in Succinic Anhydride solution. The half-life of Succinic Anhydride is very short (on the order of minutes).
