HYBRIDIZATION OF LABELED TARGETS TO MICROARRAYS

for silane and poly-L-lysine coated slides

(M. Deyholos, 21 March, 2000, based on protocols from Sigma, Pat Brown Lab, George Watts, and others)

1. Prepare a hybridization chamber by cutting a paper towel in half, then folding the paper towel to fit along the long axis of a of a 50mL screw-top tube. Wet the paper towel with 2X SSC, then drain the excess liquid, seal the tube and pre-heat to 60-65’C.

2. Combine Cy3 and Cy5 labeled targets.

3. Prepare the hybridization mixture as follows, in a 1.5mL microfuge tube

(A=for 22x22mm coverslip; B=for 22x40mm coverslip):

A B

1mL 2mL Liquid Block (Amersham RPN3601)

2mL 4mL 20X SSC

0.5mL 1mL 2% SDS

8.5mL 15mL combined labeled targets, and ddH₂O

12mL 22mL

4. Close the tube and apply a lid lock. Denature at 95’C for 2min.

5. Tansfer to ice, then spin down briefly in a microcentrifuge.

6. Preheat a blocked and denatured slide on a 65’C heat block.

7. Apply the hybridization mixture to the array, and quickly cover the liquid with a coverslip. Transfer to the pre-warmed 50mL hybridization chamber (step 1).

8. Incubate overnight at 60-65’C.

9. Wash the array with gentle shaking as follows:

2X SSC, 0.5% SDS, 65’C for 5 min.

0.5X SSC, room temp., 5min.

0.05X SSC, room temp., 5min.

10. Spin dry at low speed in a centrifuge, then scan immediately.