DNase I Treatment

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

  1. 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
  2. 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).
  3. Add 1μl of 25mM EDTA (EDTA is an exonuclease inhibitor, DNase I is a 5'exonuclease)
  4. Incubate at 65°C for 15 min to heat inactivate the DNase I; then replace on ice for 1 min.
  5. 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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