DNase I Treatment
PURPOSE
To degrade any DNA contamination in a sample of RNA. You will treat a volume of RNA that contains 1µg RNA, which can be determined using the [RNA] in µg/µl:
1µg RNA/ [RNA] µg/µl = V(µl) RNA containing 1µg
If you need to treat a larger amount of RNA, multiply each of the reaction volumes by the appropriate factor, up to 5µg RNA. Ex. If treating 2µg RNA, multiply all volumes by 2.
MATERIALS
- DNase I (Invitrogen Cat. No. 18068015)
- 10x Reaction Buffer (comes with DNase I)
- 25mM EDTA (comes with DNase I)
- DEPC-treated H2O
- Sterile 1.5ml tubes, filter tips, and pipettes (cleaned with RNaseZap)
- RNaseZap (Ambion Cat. No. 9780)
- 70% Ethanol for spraying
Notes:
- Clean bench top, pipettes, racks, with RNaseZap: spray it on everything and leave on for a few minutes, then remove carefully with Kim wipe. Then spray all with 70% Ethanol. Clean your gloves the same way.
- Perform all steps on ice, to prevent RNA degradation. Be careful not to get ice inside tubes.
- Record information for all samples and calculations as specified below.
- The amount of RNA that can be use to give a final 10μl volume is 8μl; for those samples that do not contain 1μg in the maximum of 8μl, just use 8μl. This difference will later be normalized against an internal control.
- Sample calculation shown below
PROTOCOL
- For 1μg RNA, mix the following in a 1.5ml tube, on ice:
- RNA 1 μg
- 10X Reaction Buffer 1 μl
- DNase I (1 U/μl) 1 μl
- DEPC-treated water to 10 μl
- Incubate at 37ºC for 15 min. (Note: Protocol specifies 25ºC, but DNase-treatment is often incomplete at this temperature. 37ºC is more effective).
- Add 1 μl of 25mM EDTA (EDTA is an exonuclease inhibitor, DNase I is a 5’exonuclease)
- Incubate at 65oC for 15 min to heat inactivate the DNase I; then replace on ice for 1 min.
- Collect reaction by brief centrifugation; this can be directly used for reverse transcriptase.
- Final volume: 11μl
* Order: - DEPC-treated Water
- 10X Rxn Buffer
- RNA
- DNase I
