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 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.

 

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                                     1 μg

– 10X Reaction Buffer              1 μl

– DNase I (1 U/μl)                   1 μl

– DEPC-treated water              to 10 μl

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

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