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Lab Protocol |
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Malaysia University |
PREPARATION OF SLIDES FOR MICROARRAYS
Preparation of Slides for Microarrays
Overview
DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the preparation and cleaning of the slides prior to printing the arrays.
Procedure
1. Place the slides in the slide racks and the slide racks in the chambers.
2. Prepare the Cleaning Solution.
3. Pour the Cleaning Solution into the chambers with the slides. Cover the chambers with glass lids.
4. Mix on an orbital shaker for 2 hr. Limit the exposure of the cleaned slides to air (see Hint #1).
5. Quickly transfer the racks to fresh chambers filled with ddH2O.
6. Rinse the slides vigorously by plunging the racks repeatedly in the ddH2O.
7. Repeat the rinses four times with fresh ddH2O each time (see Hint #2).
8. Prepare the Polylysine Solution.
9. Transfer the slides to the Polylysine Solution and shake on an orbital shaker for 15 min to 1 hr.
10. Transfer the racks to fresh chambers filled with fresh ddH2O.
11. Plunge the racks five times to rinse the slides.
12. Centrifuge the slides on microtiter plate carriers (see Hint #3) for 5 min at 500 rpm.
13. Transfer the slide racks to empty chambers with covers for transportation to the vacuum oven.
14. Dry the slide racks in a 45°C vacuum oven for 10 min (see Hint #4).
15. Store slides in closed plastic slide boxes (that do not contain rubber mats covering the bottom).
16. Before printing the arrays, confirm that the polylysine coating is not opaque and test print, hybridize, and scan the sample slides to determine the slide batch quality.
17. The slides are ready to be printed.
Solutions
Slide chamber
Shandon Lipshaw #121
Each chamber holds 350 ml 
Slide rack
Shandon Lipshaw #121 (800-245-6212)
Each rack holds 30 slides
95% Ethanol
Polylysine Solution.
Prepare in 560 ml of ddH2O
70 ml of Tissue Culture PBS
70 ml of Poly-L-Lysine (Sigma #P 8920)
Use a plastic graduated cylinder and beaker.
Cleaning Solution
Add 420 ml of 95% Ethanol
Dissolve 70 g of NaOH in 280 ml of ddH2O
If solution remains cloudy, add ddH2O until clear
Stir until completely mixed
The total volume is 700 ml
Tissue Culture PBS (1X)
0.24 g/liter Potassium Phosphate, Monobasic (KH2PO4)
0.2 g/liter KCl
1.44 g/liter Sodium Phosphate, Dibasic (Na2HPO4)
8 g/liter NaCl
Slide box (plastic only)
VWR #48443-806
BioReagents and Chemicals
Poly-L-Lysine
Slide box (plastic only)
Slide chamber
Sodium Hydroxide
Slide rack
Potassium Phosphate, Monobasic
Ethanol
Potassium Chloride
Sodium Phosphate, Dibasic
Sodium Chloride
Protocol Hints
1. Do not expose the cleaned slides to air as dust particles will interfere with the coating and printing of the arrays.
2. It is critical to remove all traces of Sodium Hydroxide and Ethanol.
3. Place paper towels below the rack to absorb liquid.
4. Vacuum is optional.
Citation and/or Web Resources
1. Lab website
