Incubate at 60C for the desired length of time calculated as follows

where LQ - transcript length (kb), Lf = desired length (kb),* and K = ~0.11 breaks/minute/kb.

* Unfortunately, experts differ on the ideal final length; it appears to be somewhere between 0.07 and 0.15 kb. A sample calculation for a 500-nucleotide transcript to be hydrolyzed to 150 nucleotides is as follows: (0.5 - 0.15)/0.11 x 0.5 X 0.15 = 24 minutes.

10- Stop the hydrolysis reaction by placing it on ice and adding 3 pi of 3 m sodium acetate (pH 6.0) and 5 pi of glacial acetic acid (10% v/v). Add 1 pi of 10 mg/ml tRNA carrier 8 pi of 3 m sodium acetate, and 250 pi of ethanol.

11. Pellet the probe and resuspend it in 50 pi of TED. Count 1 pi of a 1:100 dilution m a scintillation counter to determine the activity of the probe.

PART 5: Hybridization

Pretreatment of Sections Materials

Xylene

Proteinase K buffer 100 mM Tris 50 mM EDTA (pH 7.5) Proteinase K

Prepare fresh according to the directions given in Step 5

20x SSPE: Dissolve 175.3 g of NaCl, 27.6 g of NaH2P04.H20, and 7.4 g of EDTA in 800 ml of H20. Adjust the pH to 7.4 with NaOH and the volume to 1 liter with H20.

Triethanolamine (Sigma)

CAUTION: ethanol, NaH2P04, NaOH, triethanolamine, xylene (see Appendix 3) Procedure

1. Set up ten staining dishes containing the following ethanol solutions: 100%, 100%, 95%, 85%, 70%, 50%, 30%, 15%, distilled H20, distilled H20.

2. Remove the Paraplast from the slides as follows: Fill a staining dish with xylene, and place a small magnetic stir bar at the bottom. Set up

2x SSC

20x SSC: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml nf H O. Adjust the pH to 7.0 with NaOH and the volume or

2x SSPE

slides in a staining dish slide rack, and submerge the rack in the staining dish in xylene. Stir very slowly for 10 minutes. Repeat with fresh xylene.

The xylene can be used for at least three racks of slides.

3. Place the slides in 100% ethanol for a few mi nutes. Repeat with fresh ethanol.

4. Rehydrate the samples by subjecting the slides to an ethanol series (95%, 85%, 70%, 50%, 30%, 15%), followed by two changes of distilled H20 for 2 minutes each. Change the last distilled H20 for each rack. Do not discard the ethanol series.

5. Digest with proteinase K as follows: Warm the proteinase K buffer (100 mM Tris, 50 mM EDTA, pH 7.5) to 37°C. Prepare a fresh proteinase K solution of 5 mg/ml, and dilute this solution in warm buffer to a final concentration of 1 Jig/ml. Submerge the slide in the solution and incubate for 30 minutes at 37°C.

6. Rinse the slides twice in distilled H20.

Some protocols omit the following two steps (acetylation and salt wash) and proceed directly to the dehydration in Step 11.

7. Acetylation: Prepare 800 ml of 100 mM triethanolamine for each rack of slides.

Because triethanolamine is a thick liquid, it is easiest to measure it by weighing (12 g for 800 ml).

8. Equilibrate the rack of slides in a staining dish with 100 mM triethanolamine.

9. In a chemical fume hood, set up a second staining dish with 400 ml of 100 mM triethanolamine and a magnetic stir bar. Stir vigorously and add 1.25 ml of acetic anhydride. Stir for a further 5 seconds and then turn off stirrer and place slides in dish. Incubate for 5 minutes at room temperature.

10. Rinse the slides in 2x SSC or SSPE. Repeat.

11. Dehydrate: Subject the slides to an ethanol series: 15%, 30%, 50%, 70%, 85%, 95%, 100% for 2 minutes each. Except for 100% and 95% ethanol, use the solutions from Step 2.

12. Dry in a desiccator under vacuum (~1 hour).

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