PCR Clean-up Protocol for Microarray Spotting

Bradfield Lab Revised June 2002

Supplies:

• 96-well Millipore glass filter plate (Millipore, Part#. MAFB N0B).

• Millipore vacuum manifold (Millipore, Part#. MAVM0960R).

Reagents:

• PCR Binding Buffer: 150 mM potassium acetate (pH 4.8) and 5.3 M guanidine-HCl

• 80% ethanol.

Protocol:

1. Place Millipore glass filter plate on the vacuum manifold.

2. Pipette 180 ul of binding buffer in each well of the PCR reactions.

3. Pre-wet filters with 20 ul of binding buffer.

4. Pipette PCR reactions into wells.

5. Apply vacuum to pass solution through the wells and bind DNA to the glass fibers.

6. Wash with 200 ul of 80% ethanol.

7. Apply vacuum for 1 minute to pass the wash solution through the wells.

8. Repeat wash (steps 6 and 7) three additional times.

9. After final wash, place the Millipore plate on a collection dish and centrifuge at 3000 rpm for 5 minutes to remove residual ethanol.

10. Place Millipore plate on top of a clean 96-well microtiter plate. Align wells and tape the two plates together.

11. Add 65 ul of deionized water to each well (check pH and adjust to 7.5). Wait one minute, then centrifuge at 3000 rpm for 5 minutes to elute DNA.

12. Using the Hydra, assemble four 96 well plates into one 384 well plate.

13. Spin the 384 well plate of purified PCR products in the plate speed-vac until completely dry.

14. Resuspend the dried products with 10ul of nanopure water and vortex for 30 minutes on plate vortex.

15. Add 10ul of DMSO to the final products and store at –80C until printing.