Edge Lab Microarray Stripping Protocol

Adapted from: Wu et al., 2008. Biotechniques. 45: 573-575;

Hahnke et al., 2007. J. Biotech. 128: 1-13.
Recommended (and used) by Tonya Shearer

1. Make up enough stripping buffer solution for all arrays on the slide (8) + one
extra (9 samples total).
* NOTE: Each array should have 100U of RNase H. Concentrations of RNase vary. For
example, New England Biolabs 5U/L; Ambion 10 U/L. This concentration will affect
the volume of RNase H and water to add to the stripping buffer solution.

If using Epicentre or another company RNase H (10 U/L):
Reagent 1 mL Total 500 L 80 L 70 L 9
samples
1M Tris HCl 200 L 100 L 16 L 14 L 126 L
100mM DTT 200 L 100 L 16 L 14 L 126 L
1% BSA 60 L 30 L 4.8 L 4.2 L 37.8 L
1M MgCl
2
50 L 25 L 4 L 3.5 L 31.5 L
Nuclease-free H
2
O 480 L 235 L 29.2 L 24.3 L 218.7 L
10 U/L RNase H 10 L 10 L 10 L 10 L 10 L
each

2. Seat the gasket slide in the chamber and fill each well of the gasket slide with
stripping buffer in the drag and dispense manner.
3. Add RNase H to the stripping buffer bubble in each well of the gasket slide.
4. Slowly lower the array active side (Agilent side) DOWN onto the gasket slide,
making sure the two slides are parallel. Do not drop the microarray slide onto the
gasket slide, or you will increase the chance of sample mixing between wells.
5. Slide clamp assembly together and hand-tighten the clamp on the chamber.
6. Vertically rotate the assembled chamber to inspect/verify the placement of the
stripping solution in each well, making sure no leakage has occurred.
7. Incubate arrays in hyb oven at 37C overnight with rotation at 4 rpm.
8. Incubate Wash Buffer 2 overnight in water bath at 37C.
9. Inactivate Rnase H at 55C for 30 min.
10. Transfer to Wash Buffer 1 for 1 min.
11. Take Wash Buffer 2 out of water bath
12. Wash Buffer 2 for 1 min
13. Allow to air-dry for 1 min at RT.
14. Image slide immediately
15. Add 1X hyb buffer to each well of a gasket slide (mix up 360L 2X Hyb sln +
360L nuclease-free water per chip)
16. Incubate chip in hyb oven at 65C for 4-5 hrs (or overnight) with rotation at 10
rpm.
17. Repeat wash buffer steps 8-14 above.
18. Store slide in dark until next use.