S1?Nuclease?Protection?Assay

S1 Nuclease Protection Assay

End-label oligo (20-25 mer)

4 µl 5X T4 Kinase Buffer

5 µl [gamma-32P] ATP (7000 Ci/mmol)

10 µl water + oligo (200 ng)

1 µl T4 Kinase

1. 37 deg C for 1 hour.

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

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

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

5. Resuspend pellet in 51 µl water.

PCR-generated single-stranded DNA probe

20 µl digested plasmid (cut 5 µg of plasmid in volume of 20 µl)

51 µl labeled oligo

10 µl 10X Taq Buffer

6 µl 25 mM MgCl2

10 µl DMSO

2 µl 10 mM dNTP's

1 µl 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 µl and the salt to 0.3 M sodium acetate. Add 250 µl 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 µl of pre mix/rxn):

Pre-mix:

2 µl 5M NaCl

1.6 µl 0.5M PIPES, pH 7.0

0.2 µl 0.1M EDTA, pH 7.0

0.2 µl water

3. Add the following to your sample:

16 µl formamide

4 µl 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 µl 5M NaCl

10 µl 3M sodium acetate, pH 5.5

20 µl 0.1M zinc sulfate

2.5 µl S1 nuclease (400 U/µl)

907.5 µl water

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

8. Incubate at 37 deg C for 45 minutes.

9. Add 1 µl glycogen (20 mg/ml), 500 µl 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 µl 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.