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Page Title:
Table 2. Reagents and Volumes Required for DNase Treatment of RNA |
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 ERDC TN-DOER-R4
September 2004
DNase Treatment of RNA
Procedure
1. This protocol assumes the DNase treatment of ~100 g total RNA (at 2 g/L) collected
from 1 set of RNA extraction from 40 x 106 cells.
2. Follow the table below for the appropriate volumes of reagents needed. Depending on the
amount of RNA to be treated, scale up or down accordingly.
Table 2
Reagents and Volumes Required for DNase Treatment of RNA
Step #
Reagent
100 g RNA
200 g RNA
300 g RNA
400 g RNA
3
RNA at 1 g/L (L)
100
200
300
400
10X DNase 1 buffer (L)
20
40
60
80
DNase 1 at 1 U/L (L)
10
20
30
40
Water (L)
70
140
210
280
Total (L)
200
400
600
800
5
10X Termination Mix (L)
20
40
60
80
Keep in 1 tube
Keep in 1 tube
Split into 2 tubes
Split into 2 tubes
6
Phenol (L)
200
400
300
400
Chloroform (L)
120
240
180
240
10
Chloroform (L)
220
440
330
440
13
2M NaOAc (L)
20
40
30
40
96 percent Ethanol (L)
500
1000
750
1000
15
80 percent Ethanol (L)
100
200
300
400
3. In a 1.7-mL microcentrifuge tube, mix:
50 L total RNA (100 g)
20 L 10X DNase buffer
10 L DNase 1 (1 unit/L)
120 L RNase-free water
(Vortex, then centrifuge the tube for a few seconds to pull the contents to the bottom).
4. Incubate at 37C for 1 hour.
5. Add 20 L of 10X Termination Mix and mix well (split contents into two tubes when
treating 300 g or more).
6. Add 200 L of saturated phenol and 120 L chloroform, and vortex vigorously.
7. Centrifuge at 15,000 x g for 10 minutes.
8. Transfer top aqueous layer to a new 1.7-mL microcentrifuge tube.
9. Repeat steps 6 - 8 for another round of phenol:chloroform cleanup.
10. Add 220 L of chloroform and vortex well.
11. Centrifuge at 15,000 x g for 10 minutes.
12. Transfer top aqueous layer into a 1.7-mL microcentrifuge tube.
13. Add 20 L of 2M NaOAc and 500 L of 96 percent ethanol, and vortex well.
14. Centrifuge at 15,000 x g for 20 minutes.
15. Carefully remove and discard supernatant. Overlay pellet with 100 L of 80 percent ethanol.
16. Centrifuge at 15,000 x g for 10 minutes.
17. Carefully remove and discard supernatant. Air dry pellet for 10 minutes.
18. Dissolve the RNA pellet in 30 L of RNase-free water.
19. Pool RNA from the same treatment into a tube and mix well.
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