DNase I Treatment
To degrade any DNA contamination in a sample of RNA. You will treat a volume of RNA that contains 1�g RNA, which can be determined using the [RNA] in �g/�l:
1�g RNA/ [RNA] �g/�l = V(�l) RNA containing 1�g
If you need to treat a larger amount of RNA, multiply each of the reaction volumes by the appropriate factor, up to 5�g RNA. Ex. If treating 2�g RNA, multiply all volumes by 2.
- 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
– 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
- 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 37�C for 15 min. (Note: Protocol specifies 25�C, but DNase-treatment is often incomplete at this temperature. 37�C 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
– DNase I