http://cmgm.stanford.edu/pbrown/protocols
Poly-L-lysine Coating of Slides
1. Place 75 X 25 mm glass microscope slides (Gold Seal #3011) into 2 slide racks
2. Prepare Cleaning Solution: Dissolve 70 g NaOH pellets in 280 ml water; Add 420 ml
95% EtOH
3. Pour into Slide chamber with slides; Cover with saran wrap or glass lids; Mix on
orbital shaker 2 hours. (Refrain from exposing slides to air after this point)
4. Transfer slide racks to new chambers filled with dd water; Rinse by plunging racks up
and down.
5. Repeat rinses 4X with fresh dd water each time.
6. Prepare Poly-L-lysine solution: Add 70 ml poly-l-lysine solution (Sigma #P 8920)
and 70 ml tissue culture PBS to 560 ml water. (Use plastic beaker and graduated
cylinder.
7. Transfer slides to poly-l-lysine solution and shake 15 min to 1 hour.
8. Transfer racks to new chambers filled with dd water. Plunge up and down 5X to rinse
9. Cf. on microtiter plate carriers for 5 min at 500 rpm. (Place paper towel under slides
to absorb water)
10. Dry slides for 10 minutes 45 degrees Celsius
11. Allow slides to cure for at least one month
12. Test each batch of slides with small printing. (Hybridize with a plasmid probe and
look for spot morphology and background)
http://cmgm.stanford.edu/pbrown/protocols/
Post Processing Slides
1. Heat dd water in glass beaker to boiling
2. Rehydrate DNA on slides by holding slide above beaker (DNA side down) for 1-3
seconds
3. .Snap-dry each array (DNA side up) on a 70-80C inverted heat block for 3 seconds.
4. UV crosslink DNA to glass with Stratalinker set for 65 mJ. (Set display to "650",
which is 650 x 100 uJ.)
5. Place arrays in slide rack. Have empty slide chamber ready on orbital shaker. Be sure
the rack is bent slightly inwards in the middle, or else the slides may run into each other
while shaking.
6. Prepare BLOCKING SOLUTION: IN THE HOOD!!
-Have 3 350 ml glass chambers available (with metal tops). At this time, prepare 15ml
1M sodium borate pH= 8 in a 50 ml conical tube.
-Dissolve 6 g succinic anhydride (Aldrich #23,969-0) in approx. 325-350 mL
1-methyl-2-pyrrolidinone (Aldrich #32,863-4). Rapid addition of reagent is crucial.
-Reagents used are extremely toxic
a.Immediately after the last flake of the succinic anhydride dissolves, add the 15
mL sodium borate.
b.Immediately after sodium borate solution mixes in, pour solution into empty
slide chamber.
c.Plunge slide rack rapidly and evenly in solution. Vigorously shake up and down
for a few seconds, making sure slides never leave solution.
d.Mix on orbital shaker 15-20 min.
7. Gently plunge slide rack in water 5X
8. Plunge slide rack 5X in 95% ethanol.
9. Centrifuge slides and rack for 5 min. @ 500 rpm. Load slides quickly and evenly onto
the carriers to avoid streaking.
10. Use arrays immediately or store in slide box.
11. Dispose of harsh organic chemicals in organic waste container; Fill Slide chambers
one time with dd water to rinse; Also should be disposed of in organic waste