TITLE: Rnase A treatment 
AUTHORS: Rita   Kuo

[DESCRIPTION]
This protocol is used to remove RNA from genomic DNA. RNase A is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues. It cleaves the phosphodiester bond between the 5'-ribose of a nucleotide and the phosphate group attached to the 3'-ribose of an adjacent pyrimidine nucleotide. The resulting 2', 3'-cyclic phosphate is hydrolyzed to the corresponding 3'-nucleoside phosphate.

[STEPS]
SECTION: Rnase A treatment
1. Bring up the sample volume to 200 ul using TE buffer (pH 8.0)
SECTION: Rnase A treatment
2. Add 10 ul of RNase A
SECTION: Rnase A treatment
3. Incubate 37°C for 1 hour.
SECTION: Sample QC
6. Run gel 10-15 minutes. If RNA is visible on the gel, add another 10 ul of RNase A and repeat the incubation step, otherwise proceed ethanol precipitation. 

See the Lane D on following figure for an example of RNA contaminated sample. 

  

SECTION: Ethanol Precipitation
28. Note: Prepare 75 % ethanol and place it on ice beforehand.  

Add 1/10 volume of 3 M sodium acetate to RNase A treated DNA 
SECTION: Ethanol Precipitation
28. Add 2.5 X volumes of 100% ethanol
SECTION: Ethanol Precipitation
28. Gently mix and spin down the sample at 5000 rpm for 1 min. 
SECTION: Ethanol Precipitation
28. Place at -80°C for 30 minutes (or -20°C for overnight). Make sure the tube is -80 °C freezer safe. 
SECTION: Ethanol Precipitation
28. Centrifuge at full speed (13000rpm), at 4°C for 20 minutes to pellet DNA
SECTION: Ethanol Precipitation
28. Pour supernatant and wash pellet with freshly made 75 % cold ethanol.
SECTION: Ethanol Precipitation
28. Quick spin sample at 4°C for 30 seconds
SECTION: Ethanol Precipitation
28. Carefully remove ethanol with pipet tips
SECTION: Ethanol Precipitation
28. Dry the pellet in room temperature
SECTION: Ethanol Precipitation
28. Suspend the pellet with 30 ul (or appropriate volume) of TE
SECTION: Ethanol Precipitation
28. Use Nanodrop for qualification and Qubit for quantification. The ratio of 260/280 should be 1.8 for DNA and a typical spectral pattern of DNA should look like this:

  

SECTION: Sample QC
5. Load about 25ng/5 ul of sample on an agarose gel and use untreated sample as a control.
SECTION: Sample QC
4. NOTE: Buffers and enzyme mix in the sample will affect Spectrophotometer reads. It's better to use gel to verify if sample still contains RNA. 

Make 0.8 % agarose gel.
SECTION: Ethanol Precipitation
7. 

Add 1/10 volume of 3 M sodium acetate to RNase A treated DNA.
SECTION: Ethanol Precipitation
8. Add 2.5 X volumes of 100% ethanol
SECTION: Ethanol Precipitation
9. Gently mix and place it at -80°C for 30 minutes (or -20°C for overnight).

NOTE: Prepare 75 % ethanol and place it on ice before the next step. 
SECTION: Ethanol Precipitation
10. Centrifuge at full speed (13000rpm), at 4°C for 20 minutes to pellet DNA.

SECTION: Ethanol Precipitation
11. Pour supernatant and wash pellet with freshly made 75 % cold ethanol.
SECTION: Ethanol Precipitation
12. Centrifuge at top speed at 4°C for 1 minutes.
SECTION: Ethanol Precipitation
13. Carefully remove ethanol with pipet tips
SECTION: Ethanol Precipitation
14. Suspend the DNA pellet with 30 ul (or appropriate volume) of nuclease free water .
SECTION: Ethanol Precipitation
15. Use Nanodrop for qualification and Qubit for quantification. The ratio of 260/280 should be 1.8 for DNA and a typical spectral pattern of DNA should look like this: