RNase H Digestion of RNAs

  1. Dry down 10 µg RNA and 300 ng oligo in speed vac.
  2. Resuspend pellet in 10 µL 1X Hyb Mix.
  3. Head at 68ºC for 10 minutes. Cool slowly to 30ºC. Pulse spin down.
  4. Add 10 µL 2X RNase H Buffer. Mix.
  5. Incubate at 30ºC for 60 minutes.
  6. Add 130 µL Stop Mix.
  7. Phenol/Chloroform extract
         Add 1 vol. phenol/chloroform
         Vortex well.
         Spin down 2 minutes in room temperature microfuge.
         Remove top layer to new tube.
  8. Chloroform extract
         Add 1 vol. chloroform
         Vortex well.
         Spin down 2 minutes in room temperature microfuge.
         Remove top layer to new tube.
  9. Add 375 µL 100% ethanol. Freeze at -80ºC. Spin down 10 minutes in room temperature microfuge.
  10. Wash pellet with 70% ethanol.
  11. Resuspend in 10 µL loading dye.
  12. Heat at 100ºC for 3 minutes immediately before loading.

Solutions
All solutions and reagents MUST be prepared using DEPC'd H2O. All solutions stored at -20ºC.

10X Hyb Mix

For 10 mL:

0.25 M Tris, pH 7.5
10 mM EDTA
0.5 M NaCl
2.5 mL 1 M Tris, pH 7.5
0.2 mL 0.5 M EDTA
1 mL 5 M NaCl
6.3 mL DEPC'd H2O

2X RNase H Buffer

For 10 mL:

40 mM Tris, pH 7.5
20 mM MgCl2
100 mM NaCl
2 mM DTT
60 µg/mL BSA 		0.4 mL 1 M Tris, pH 7.5
0.2 mL 1 M MgCl2
0.2 mL 5 M NaCl
40 µL 0.5 M DTT
240 µL 2.5 µg/mL BSA
9 mL DEPC'd H2O
Add 0.5 µL RNase H to 9.5 µL buffer fore each reaction
immediately prior to use.

Stop Mix

For 20 mL:
.04 mg/mL tRNA
20 mM EDTA
300 mM NaOAc		 16 µL 50 mg/mL tRNA
0.8 mL 0.5 M EDTA
2 mL 3 M NaOAc
17.2 mL DEPC'd H2O

Formamide Loading Dye

For 1 mL

100% deionized formamide
5 mM EDTA
0.25% bromophenol blue
0.25% xylene cyanol 		1 mL deionized formamide
10 µL 0.5 M EDTA
20 % micro;L 1% bromophenol blue
20 %micro;L 1% xylene cyanol


This site maintained by Anne Webb amwebb@email.arizona.edu
©Copyright 1998; All rights reserved.
Last updated 8/22/06