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S1 Nuclease Protection Assay

End-label oligo (20-25 mer)

4 l 5X T4 Kinase Buffer
5 l [gamma-32P] ATP (7000 Ci/mmol)
10 l water + oligo (200 ng)
1 l T4 Kinase

1. 37 deg C for 1 hour.

2. Heat inactivate at 75 deg C for 10 minutes.

3. Add 1 l glycogen (20 mg/ml), 21 l 4M ammonium acetate, 90 l EtOH and ppt.

4. Resuspend pellet in 26 l water, and add 26 l 4M ammonium acetate, 110 l EtOH and ppt. again.

5. Resuspend pellet in 51 l water.

PCR-generated single-stranded DNA probe

20 l digested plasmid (cut 5 g of plasmid in volume of 20 l)
51 l labeled oligo
10 l 10X Taq Buffer
6 l 25 mM MgCl2
10 l DMSO
2 l 10 mM dNTP's
1 l Taq polymerase

14 cycles of PCR

Run on 6-8% polyacrylamide/urea gel

Cut out probe and elute

S1 mapping

1. Coprecipitate RNA + 1E4 cpm of probe by adjusting final volume to 100 l and the salt to 0.3 M sodium acetate. Add 250 l EtOH and ppt.

2. Wash pellet with 70% EtOH and dry for 2 minutes in speed vac.
DO NOT OVER DRY.

For hybridization, make pre-mix of salts in a seperate tube (will use 4 l of pre mix/rxn):
Pre-mix:
2 l 5M NaCl
1.6 l 0.5M PIPES, pH 7.0
0.2 l 0.1M EDTA, pH 7.0
0.2 l water

3. Add the following to your sample:
16 l formamide
4 l pre-mix salts

4. Vortex vigorously.

5. Incubate at 100 deg C for 3 minutes.

6. Transfer immediately to 58 deg C and incubate overnight.

S1 digestion buffer (final concentration):
300 mM NaCl
30 mM sodium acetate, pH 5.5
2 mM zinc sulfate
1000 units/ml S1 nuclease

For 1 ml of digestion buffer:
60 l 5M NaCl
10 l 3M sodium acetate, pH 5.5
20 l 0.1M zinc sulfate
2.5 l S1 nuclease (400 U/l)
907.5 l water

7. Add 200 l of S1 digestion buffer to sample.

8. Incubate at 37 deg C for 45 minutes.

9. Add 1 l glycogen (20 mg/ml), 500 l EtOH and precipitate on ice for 15 minutes.

10. Spin for 30 minutes at 4 deg C.

11. Wash with 70% EtOH.

12. Speedvac and add 5 l formamide loading dye (5X TBE, 45% formamide, 0.25% bromphenol blue). Boil samples for 3 minutes and place on ice.

13. Run on 6-8% acrylamide gel, dry gel, and expose to film.

 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Aug 29, 2001


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