Probe Synthesis Materials

Components of the DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals) or DIG-labeling components Template DNA Phenol-chloroform

Reagents for in vitro transcription and DIG labeling RNase inhibitor RNase-free DNase lOx DNase buffer (100 ml) 40 ml of 1 M Tris (pH 7.5) 6 ml of MgCl2 54 ml of distilled H20 Adjust pH to 7.5. Equipment for agarose gel electrophoresis of RNA EDTA (0.5 M) LiCl (4 M) Ethanol

CAUTION: chloroform, ethanol, LiCl, MgCl2, phenol, Tris (see Appendix 3)

Procedure

1. Linearize template plasmid by restriction digest. Use only enzymes that create a 5' overhang to avoid spurious initiation of RNA transcription. Extract with phenol-chloroform and precipitate. Dissolve the probe m RNase-free distilled H20.

2. Assemble run-off transcription reaction at room temperature:

1 mg/pl BSA 2 pi lOx transcription buffer 2 pi

10% Triton X-100 2 pi lOx DIG RNA labeling mix 2 pi

RNase inhibitor 1 pi template (total of 1 pg) up to 9 pi

RNA polymerase (T7, T3, SP6) 2 pi distilled H20 to 20 pi

Incubate the reaction for 2 hours at 37°C.

3. DNase treatment. Add to the transcription reaction:

RNase inhibitor 1 pi lOx DNase buffer 5 pi

RNase-free distilled H2G 2 pi

RNase-free DNase I (23 units) 1 pi

Incubate the reaction for 15 minutes at 37°C.

4. Stop the reaction and precipitate by adding:

100% ethanol (chilled) 180 pi

Store overnight at -80°C.

5. The following day, centrifuge for 15 minutes in a microcentrifuge and discard the supernatant.

6. Wash the pellet with 200 pi of chilled 100% ethanol. Centrifuge for 5 minutes.

7. Dry and redissolve the pellet in 95 pi of RNase-free distilled H20.

8. Add 1 pi of RNase inhibitor and incubate for 30 minutes at 37°C.

9. Run 10 p.1 of the probe on an RNA gel to check size and integrity of the transcript.

10. Quantify the probe according to the manufacturer's protocol (Roche Molecular Biochemicals). About 20-70 ng of probe is required per slide.

Pretreatment of Sections Materials

Hemo-De NaCl

PBS (see page 198 for PBS recipe) 2x SSC (see page 188 for SSC recipe)

Proteinase K (10 mg/ml in 0.1 M Tris-Cl [pH 7.5], 0.05 M EDTA

[pH 8.0]) Acetic anhydride (0.5% v/v) Triethanolamine (0.1 M, pH 8.0) Fixative (see page 203)

CAUTION: acetic anhydride, ethanol, formamide, HCl, methanol, triethanolamine (see Appendix 3)

Procedure

1. Place the slides in a rack and move them through the following series:

Hemo-De

10 minutes

Hemo-De

10 minutes

methanol

15 minutes

100% ethanol

1 minute

100% ethanol

1 minute

95% ethanol

1 minute

85% ethanol/0.85% NaCl

1 minute

70% ethanol/0.85% NaCl

1 minute

50% ethanol/0.85% NaCl

1 minute

30% ethanol/0.85% NaCl

1 minute

0.85% NaCl

2 minutes

2. Transfer the slides to lx PBS for 2 minutes and then to 0.2 M HC1 for 20 minutes.

3. Rinse with distilled H O and immerse in 2x SSC for 20 minutes.

4. Rinse with distilled H O and then transfer to 10 pg/ml proteinase K {in 0.1 M Tris-HCl [pi [ 7.5], 0.05 M EDTA [pH 8.0]) for 30 minutes at 37°C. Warm the buffer to 37°C before adding the proteinase K.

5. Wash the slides by placing them in lx PBS for 2 minutes and then fix them for 10 minutes in fixative (see page 203 for fixative recipe).

6. Transfer the slides to 0.5% (v/v) acetic anhydride in 0.1 M tri-ethanolamine (pH 8.0) for 10 minutes.

Add acetic anhydride one drop at a time while stirring the tri-ethanolamine solution on a magnetic stirrer.

7. Wash in lx PBS for 2 minutes.

9. Dehydrate the slides by moving them through the solutions listed in Step 1, but in reverse order. Reuse all ethanol solutions except 100% ethanol.

10. Store the slides overnight at 4°C in a container with 100% ethanol covering the bottom or go immediately to pretreatment for hybridization.

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