TITLE: DNase treatment with Invitrogen kit
AUTHORS: Anna JSzékely

[DESCRIPTION]
We use this protocol to remove DNA from DNA/RNA mix isolated with Easy-DNA Kit (see Simultaneous DNA/RNA extraction for filtered water samples protocol).

[BEFORE_START]
 
Clean all surfaces (bench, pipettes, racks) before starting to ensure removal of contaminants. To do this wipe sequentially with a combination of (1) MQ, (2) 70% ethanol and (3) RNAse Away.
Get ice
Take out from the freezer and thaw: 10xBuffer, EDTA (included in kit)
Get samples from -80ºC
Label tubes or strips, one for each sample and one as negative control.
Leave DNase in freezer until use. DNase is sensitive to physical denaturation, therefore never vortex it!
 You can also apply 10 minutes UV exposure in the UV-box for pipettes, tips, racks, etc. 
 
 

[GUIDELINES]
SAFETY: One of the kit components contains the following chemicals:
8-10% of human urine (potential biohazard)
0.1-0.3% bovine serum albumin (harmful)
≤0.09% sodium azid (very toxic).
As both the amount and the concentration of the very toxic compound (sodium azid) is very low, the risk is also low.
Avoid contact with eyes, skin and other mucous membranes. Wash thoroughly after handling. Avoid breathing vapor/mist/spray.
 
QUALITY ASSURANCE: 
Controls: Run a negative DNase treatment sample for every batch of DNase treatment to control for potential contaminations of reagents or during the treatment procedure.
Kit properties: Always write down the LOT number of the kit you use. Avoid using expired kits or reagents.
Test PCR for treatment: always perform test PCR (step 10-11.) to confirm the success of the DNase treatment. 
VERY IMPORTANT: RNA is much more sensitive to degradation than DNA, therefore contamination with RNase has to be avoided (the main source is your own skin) and the samples have to be kept on ice all the time to slow degradation. The following steps help avoid degradation before and during RNA work:
Before starting to work clean bench, pipettes and the racks with MQ water (against DNA and RNA contamination), 70% ethanol (desinfection) and RNaseAway spray.
Use only nuclease-free filter tips and tubes.
Keep thawed RNA samples on ice all the time.
Use new gloves to touch samples and RNase-free pipettes and racks (cleaned in point 1). If you touch anything else (for example freezer handle), immediately change gloves. Change gloves regularly anyway during work with RNA, at least after every few steps. Before starting to work, spray RnaseAway on your gloves.
Special care should be applied until RNA is completely transcribed to DNA (end of RT process).

[STEPS]
SECTION: Mix preparation
1. Prepare incubation mix (for few samples) and continue at step 5:
 
 
 
There are no exact indications for the amount of sample to use but I usually start with the maximum amount (8 µl) and reduce it only for the samples were the test PCR is positive.
 
For more samples prepare master mix (MM) of 10x buffer and DNase I (never vortex!), put back to freezer after preparing MM).
 
 
SECTION: Mix preparation
2. Add RNAse-free water to tubes if needed.
Calculate the amount like this: (8μL - amount of sample) * n * 1.15
For higher DNA/RNA content samples use less than 8μL.
 
SECTION: Mix preparation
3. Mix gently and spin MM, and distribute in labelled PCR tubes/strips.
SECTION: Mix preparation
4. Add 1-8 μL of DNA/RNA sample or diluted sample to each tube, keep tubes on ice.
SECTION: Mix preparation
5. Mix gently and spin tubes.
SECTION: Digestion
6. Incubate tubes in PCR machine at 25ºC for 15’ (DNA digestion).
15 min
SECTION: Inactivation
7. Move tubes back to ice and add 1 μL EDTA to each, vortex and spin.
SECTION: Inactivation
8. Incubate tubes in PCR machine at 65ºC for 10’ (DNase inactivation).
10 min
SECTION: Test PCR
10. Perform test PCR. Always use a positive control and a negative control!
 
We do everything the same way as we will generate the amplicons for Illumina sequencing with the trancribed cDNAsing except that we do only one longer PCR. We also use only half amount of template (0.5 μl) as the cDNA will also be diluted during the RT reaction.
 
Primers:
Illumina adapter-N4-341F:
3’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACGGGNGGCWGCAG-5’
Illumina adapter-805NR:
3’-AGACGTGTGCTCTTCCGATCTGACTACNVGGGTATCTAATCC-5’
 
PCR reactions
 
 
 
 
DNase-test PCR program
 
 
 
SECTION: Test PCR
11. Check PCR products in 1% Agarose gel. 
If there is product only in the postivie control but not in the others, continue with RT protocol.
If there is product in a sample, repeat the DNAse treatment for that product with less template or diluted template.
SECTION: Inactivation
9. Spin and store tubes at -20ºC until next step.