P-FPMI-53 - P-FPMI-53

Type
hybridization
Description
1.Follow instructions in Two-Colour Nucleic Acid Microarray Toolkit Handbook (Invitrogen).

2.Incubate slides in Prehybridization buffer (prewarmed to 42•C) for 5-45 min.

3.Rinse slides in de-ionized water at room temperature. Rinse all slides together (in grey slide-rack provided) in green wash tanks. Submerge microarrays and dip up and down in de-ionized water 20 X. Add fresh, de-ionized water and repeat 3X.

4.Dry the slides, in slide-rack, under a stream of clean, filtered compressed air.

5.Lie slides down individually on a clean, dry, lint-free cloth in the provided hybridization chamber. Add clean Lifterslip to each slide.

6.Preheat hybridization solution at 42•C. Add 42 ul hybridization solution, 1 ul Salmon sperm DNA, 10mg/ml) and 42 ul Labeled Target DNA.

7.Heat at 95• for 5 min. Move tubes to 65•C until use.

8.Slowly add 80 ul hybridization mixture to 1 side of the lifterslip allowing the solution to be pulled via capillary action across the array surface.

9.Incubate at 42•C overnight in the closed hybridization chamber. Add 10 ml de-ionized water to the base of the hybridization chamber prior to overnight incubation.

10.Preheat Solution 1. Use blue forceps to clasp each slide being careful not to clasp liferslip to the microarray. Use squirt bottle to apply solution 1 to each array in turn. Squirt at least 5ml worth of solution 1 to each side of the slide, being careful not to let either side dry out. Put microarray in slide rack in preheated solution 1 in wash tanks. Process each slide.

11.Dip slide rack 10X and incubate for 10 minutes at 42•C. Add fresh solution 1 to wash tanks and repeat.

12.Dip slide rack 10X and incubate in solution 2 at room temperature for 10 minutes.

13.Fill 3 wash tanks with solution 3, at room temperature, dip 10X each and incubate in succession for 1 minute each.

14.Add 4 slides to 25ml of blocking solution in a clean slide mailer. Incubate for 2 minutes. Remove each slide and place on clean, damp, cloth in hybridization chamber. Add 80 ul RLS particle solution (30 ul Ag particle, 30 ul Au particle, 30 ul diluent, freshly prepared) to each slide. Apply lifter slip to one side and allow the slip to fall onto the remainder of the slide, gently distributing the RLS particles in solution over the remainder of the slides. Repeat for the rest of the slides.

15.Incubate in hybridization chamber for 1 hour.

16.Use blue forceps to clasp each slide being careful not to clasp liferslip to the microarray. Use squirt bottle to apply solution 2 (room temperature) to each array in turn. Squirt at least 10 ml worth of solution 2 to each side of the slide, being careful not to let either side dry out. Put microarray in slide rack containing solution 3 in wash tanks. Process each slide.

17.Fill 3 wash tanks with solution 3, at room temperature, dip 10X and incubate in succession for 1 minute each.

18.Rinse slides in de-ionized water at room temperature. Submerge microarrays up and down in de-ionized water 20 X. Add fresh, de-ionized water and repeat 3X.

19.Dry the slides, all together, under a stream of clean, filtered compressed air.

20.Clasp individual slide with blue forceps and submerge in Archiving solution. Do not submerge the tips of the forceps.

21.Lean slide against solid support to dry. Dry on clean, lint-free cloth. Apply pressure to the slides periodically to ensure archive solution does not pool and dry to the bottom of the slides.

22.When dry, scan slides or put into a lint-free container to protect from dust.

Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
All experiments using protocol P-FPMI-53: (E-FPMI-2, E-FPMI-23, E-FPMI-3)