DNase treatment of RNA prep:
Materials Needed:
10ul DNaseI and 10ul DNase 10X buffer per sample
Phenol/Chloroform (5:1) (Ambion)
100% EtOH.
3M NaOAc (sodium acetate) pH 5.7 (or 5.2)
Microcentrifuge (4oC)
P-1000, P-200, P-20 pipettors.
Procedure:
1. Make up master mix of DNase I and 10X buffer for all samples to be
treated. For example, if there are 10 samples (80ul volume), then make a
master mix contaning 10ul of both DNase I and 10X buffer for each
sample. Make slightly more than is needed to correct for pipetting error. In
the sample above, make up a mix for 10.5 reactions = 105 ul 10X Buffer +
105ul DNase I.
2. Pipette appropriate volume master mix into each sample. In the example
above, add 20ul master mix to each 80ul sample (1X final concentration).
3. Place tubes @37oC (heat block or water bath) for 30’.
4. Add equal volume of phenol/chloroform (5:1) to each sample (100ul for
example above).
5. Shake vigorously or vortex for 15 seconds, then incubate 1’ @RT.
6. Centrifuge @13K rpm, 10’, RT. Label fresh set of tubes.
7. Carefully remove top layer (aqueous phase) and pipet into fresh tubes.
8. Add 1/10 volume NaOAc and 3 volumes EtOH to each sample (a master
mix can be prepared ahead of time if desired).
9. Place samples on ice (ice/H2O slurry) for 1-2hrs. Alternatively, the
samples can precipitate @ -20oC overnight (this gives much bigger
pellets.)
10. Centrifuge @13K rpm, 15’, 4oC.
11. Carefully remove supernatant.
12. Wash pellet with appropriate volume 75% EtOH
13. Centrifuge @ 10k rpm, 5’, 4oC. Carefully remove all traces of super natant
wash being careful not to disturb pellet.
14. Let pellet air dry for ~5’.
15. Resuspend pellet in appropriate volume sterile H2O. RNA can be stored
at -20oC (short term) or -80oC (long term).