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 1μg
- 10X Reaction Buffer 1μl
- DNase I (1 U/μl) 1μl
- DEPC-treated water to 10μl
- 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 1μl of 25mM EDTA (EDTA is an exonuclease inhibitor, DNase I is a 5'exonuclease)
- Incubate at 65°C 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
Tube | Condition | [RNA] μg/μl | Volume for 1μg | Volume (μl) for up to 8μl | DEPC-treated H2O to 8μl | 10X Reaction Buffer (μl) | DNase I (μl) | Genotype |
---|---|---|---|---|---|---|---|---|
1 | Condition | 0.179 | 5.59 | 5.59 | 2.41 | 1.00 | 1.00 | +/+ |
2 | Condition | 0.184 | 5.43 | 5.43 | 2.57 | 1.00 | 1.00 | +/+ |
3 | Condition | 0.149 | 6.71 | 6.71 | 1.29 | 1.00 | 1.00 | +/+ |
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