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Purpose: After spotting cDNAs onto glass slides, the slides
must be treated to bind the DNAs to the coating and to denature
the DNAs so they can hybridize to the fluorescently labeled probe
DNA. During the blocking step the array will disappear so be sure
that the boundary of the array has been marked with a diamond pin
as described in microarray construction. During the process,
the exposed aldehyde groups by are blocked by reducing them to alcohol
groups using sodium borohydride (NaBH4).
Record print ID and slide ID numbers: _____________________________
Person name and date: _____________________________
Check off each step as it is completed:
_____ The slides should have previously been Hydrated and UV
crosslinked just after printing. Check the log and IDs of the
printed slides to be sure that this was done.
_____ During the blocking step the array will disappear so be
sure that the boundary of the array has been marked with a diamond
pin as described in microarray construction.
_____ Prepare the following reagents and supplies: We use
Wheaton #900200 glass staining dishes (need at least 5) to do the
blocking of the slides. These hold 10 slides each but we only put
6 slides in order to get the maximum blocking. Also need a small
magnetic stirring bar (1/2 x 5/16 inch) and a regular stirring plate.
450 mls 0.2% SDS (200 mls in 2 separate dishes) (make by mixing
30 mls 3% SDS plus 420 mls water)
Wire holders for racks
2 Wheaton slide staining dishes with nano pure water to rinse
slides well
1 2L microwavable plastic beaker with 1 L water
____1. Rinse all glassware with EtOH and dry well.
____2. Set up everything on bench. Place aldehyde, silylated
slides that have been spotted in glass staining rack (not more than
6 in each glass rack). Place stir bar in staining dish. Place rack
of spotted slides in dish. Put on stir plate and verify stir bar
will not hit the slides.
____3. Start boiling 1 liter water in 2 liter plastic beaker in
microwave (8 minutes at high power)
____4. Prepare fresh: 200 mls 0.25% NaBH4 solution (0.5g
NaBH4, 150 mls 1X PBS, 50 mls 95%EtOH) (sodium borohydride,
Aldrich #48,088-6, Lot # _______stored under
desiccation).
____5. Put slides through the follow series with vigorous shaking
by orbital shaker or stir bar on stirring plate:
___ 1. 0.2% SDS 2 min
___ 2. water rinse 2 min
___ 3. water rinse 2 min
___ 4. 0.25% NaBH4 solution 5 - 10 min
___ 5. 95c water 2 min
___ 6. 0.2% SDS 2 min
___ 7. water rinse 2 min
___ 8. water rinse 2 min
____6. Transfer to metal racks. Keep slides under water until all
are collected.
____7. Place rack of slides on top of styrofoam pad (prevents metal
rack from bending down in the middle). Spin for 2 min at 500 rpm
to dry.
____8. Store in a slide rack in a dust-free container.
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