DNase treatment with Invitrogen kit

Prepare incubation mix (for few samples) and continue at step 5:

A	B
	Amount (μL)
DEPC-treated water (not MQ)	0-7
10xbuffer	1
DNase I	1
Sample or diluted sample	1-8
Total	10

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).

A	B	C
	For 1 sample (μL)	For n sample (μL)
10xbuffer	1	1n1.15 (e.g. for 20 samples, add 23 μL)
DNase I	1	1n1.15

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.

Mix gently and spin MM, and distribute in labelled PCR tubes/strips.

Add 1-8 μL of DNA/RNA sample or diluted sample to each tube, keep tubes on ice.

Mix gently and spin tubes.

Incubate tubes in PCR machine at 25ºC for 15’ (DNA digestion).

0h 15m 0s

Move tubes back to ice and add 1 μL EDTA to each, vortex and spin.

Incubate tubes in PCR machine at 65ºC for 10’ (DNase inactivation).

0h 10m 0s

Spin and store tubes at -20ºC until next step.

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

A	B	C	D	E	F
Components	Working conc.	Final conc.	1 reaction (20 µl)	(N) reactions
5xQ5 Reaction Buffer	5X	1X	4 µl
Forward Primer (illu-ada-341F)	10 µM	0.25 µM	0.5 µl
Reverse Primer (illu-ada-805NR)	10 µM	0.25 µM	0.5 µl
dNTPs	2 mM	200 µM	2 µl
Q5 HF DNA polymerase	2 U/µl	0.02 U/µl	0.2 µl
Template DNA			0.5 µl
Nuclease-Free water			12.3 µl
∑			20 µl

DNase-test PCR program

A	B	C
STEP	TEMP.	TIME
Initial Denaturation	98oC	30 seconds
35 cycles	98oC	10 seconds
48oC	30 seconds
72oC	30 seconds/kb
Final Extension	72oC	2 minutes
Hold	6oC	∞

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.