poly-L-lysine (0.1% w/v) 35 ml PBS 35 ml H2O 280 ml 350 ml
Method
1. Place slides into 50 slide racks and place racks in glass tanks with 500 ml of cleaning solution. Gold Seal Slides are highly recommended, as they have been found to have consistently low levels of autofluorescence.
It is important to wear powder free gloves when handling the slides. Change gloves frequently, as random contact with skin and surfaces transfers grease to the gloves.
2. Place tanks on platform shaker for two hours at 60 rpm.
3. Pour out cleaning solution and wash in H2O for three minutes. Repeat wash four times.
4. Transfer slides to 30 slide plastic racks and place into small plastic boxes for coating.
5. Submerge slides in 200 ml poly-L-lysine solution per box.
6. Place slide boxes on platform shaker for one hour at 60 rpm.
7. Rinse slides three times with H2O.
8. Submerge slides in H2O for one minute.
9. Spin slides in centrifuge for two minutes at 400 xg and dry slide boxes used for coating.
10. Place slides back into slide box used for coating and let stand overnight before transferring to new slide box for storage.
This allows the coating to dry before handling.
11. Allow slides to age for two weeks on the bench, in a new slide box, before printing on them. The coating dries slowly, becoming more hydrophobic with time.
Slide boxes used for long term storage should be plastic and free of cork lining. The glue used to affix the cork will leach out over time and give slides stored in these types of boxes a greasy film that has a high degree of autofluorescence. Clean all glassware and racks used for slide cleaning and coating with highly purified H2O only. Do not use detergent.
2.9. SLIDE BLOCKING
At the end of the print, slides are removed from the printer, labeled with the print identifier and the slide number by writing on the edge of the slide with a diamond scribe and placed in a dust free slide box to age for one week.
It is useful to etch a line, which outlines the printed area of the slide, onto the first slide. This serves as a guide to locate the area after the slides have been processed, and the salt spots are washed off.
1. Place slides, printed face up, in casserole dish and cover with cling wrap. Expose slides to a 450mJ dose of ultraviolet irradiation in the Stratalinker.
Slides should have been aged at ambient temperature in a closed slide box for one week prior to blocking.
2. Transfer slides to a 30 slide stainless steel rack and place rack into a small glass tank.
3. Dissolve 6.0g succinic anhydride in 325ml 1-methyl-2-pyrrolidinone in a glass beaker by stirring with a stir bar.
Nitrile gloves should be worn and work carried out in a chemical fume hood while handling 1-methyl-2-pyrrolidinone (a teratogen).
4. Add 25ml 1M sodium borate buffer (pH 8.0) to the beaker. Allow the solution to mix for a few seconds, then pour rapidly into glass tank with slides.
Succinic anhydride hydrolyzes quite rapidly once the aqueous buffer solution is added. To obtain quantitative passivation of the poly-L-lysine coating, it is critical that the reactive solution be brought in contact with the slides as quickly as possible.
5. Place the glass tank on a platform shaker in a fume hood for 20 minutes.
Small particulates resulting from precipitation of reaction products will be visible in the fluid.
6. While the slides are incubating on the shaker, prepare a boiling H2O bath to denature the DNA on the slides.
7. After the slides have incubated for 20 minutes, transfer them into the boiling H2O bath. Immediately turn off the heating element after submerging the slides in the bath. Allow slides to stand in the H2O bath for 2minutes.
8. Transfer the slides into a glass tank filled with 100% ethanol and incubate for 4 minutes.
9. Remove the slides and centrifuge at 400 rpm for 3 minutes in a horizontal microtiter plate rotor to dry the slides.
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