Dnase 1 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

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s