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David Weaver et al.
4 Notes 1. Affymetrix has discontinued support of the GCOS software, and the only available operating system is the AGCC software (http://www.affymetrix.com/esearch/search.jsp?pd=13142 9&N=4294967292). The researcher will have to use the AGCC software to do experiments with the Affymetrix system. 2. Following preparation of the solution, the pH normally does not need much adjustment. To avoid a sudden pH change, use either 1 N HCl to lower the pH or 1 N NaOH to increase the pH. 3. Do not autoclave the solution. If the solution turns yellow with time, discard the solution. 4. Affymetrix sells premade Hybridization, Wash and Stain kits (catalog number 900720) (Affymetrix) with all of the necessary components for the hybridization, array post-hybridization washing, and staining. 5. Frozen stocks of 20× Eukaryotic Hybridization Controls are heated to 65 °C for 5 min to completely resuspend the cRNA before dispensing. 6. It is important to allow the arrays to be at room temperature prior to use. The rubber septa (on the back of the array for loading) need to be equilibrated to room temperature; otherwise, they may be prone to cracking which can result in leaks. 7. It is recommended that lock caps be used on the microcentrifuge tubes during the high heat incubation to ensure the lids do not open as a result of the increased temperature. 8. Make up a small volume of 1× hybridization buffer by making a 1:1 dilution of the 2× hybridization buffer. The 200 μL volume will not completely fill the array. Move the array, while looking in the array window, to ensure the array is completely wetted. The solution should be shielded from light and stored at 2–8 °C. Another option is to make a 1× array holding buffer: 100 mM MES, 1 M [Na+], 0.01 % Tween-20. For 100 mL, combine 8.3 mL of 12× MES stock solution, 18.5 mL of 5 M NaCl (RNase-free, DNase-free), 0.1 mL of 10 % Tween-20, and 73.1 mL ultrapure water. Mix and filter the solution through a 0.2 μm filter. The solution should be shielded from light and stored at 2–8 °C. 9. The scanner uses a laser and has a safety interlock system; however, users should always be aware of the hazardous nature of laser light. It is important to let the scanner laser warm up for at least 10 min prior to scanning for the laser to stabilize. 10. As mentioned previously, our experiments were carried out with the GCOS operating system. This software had the capa-
