DNase I Treatment Of Total RNA

Reagents

1 U/ul DNase I from Epicentre Technologies

10X DNase I buffer （200 mM Tris, pH 8.4, 20 mM MgCl2, 500 mM KCl）

For 1ml:200 mM Tris, pH 8.4200 ul 1M Tris, pH 8.4500 ul 1M KCl20 ul 1 M MgCl2280 ul DEPC water25 mM EDTARNeasy Kit from Qiagen

According to Invitrogen, reactions can be set up in a minimum of 10 ul and scaled up if larger volumes of RNA need to be treated. Treatments can range from 22-37°C for 15-30 minutes using 0.1-3 units of DNase I.

Invitrogen's suggested mix:

1 ug RNA1 ul DNaseI （1 U/ul）1 ul 10X DNaseI bufferDEPC water to 10 ul

Example of a scaled up version for a 40 ul reaction

100 ug Total RNA

4 ul DNase I （1 U/ul）

4 ul 10X DNaseI buffer

DEPC water to 40 ul

Prepare your reaction and mix well.

Incubate 37°C for 30 minutes. Stop reaction by adding 25 mM EDTA, pH 8.0; add 1 ul per 10 ul of reaction volume.

Perform cleanup with an RNeasy column. Be sure to add 2-ME to RLT and ethanol to RPE buffers. Steps have been modified （see **） according to Qiagen and Invitrogen.

Bring RNA to 100 ul with DEPC water

Add 350 ul RLT, mix well

Add 250 ul ethanol, mix well

Apply to column, spin >10,000 rpm 15 sec

Take eluate and place back onto same column, spin >10,000 rpm 15 sec

Discard eluate; Add 700 ul RW1 buffer （**）， spin >10,000 rpm 15 sec

Move column to new 2 ml collection tube

Add 500 ul RPE buffer, spin >10,000 rpm 15 sec

Discard flow through; Add 500 ul RPE buffer, spin 10,000 rpm 1 min

Discard flow through and spin column for 30-60s at maximum speed

Move column to 1.5 ml tube, add 30 ul DEPC water, incubate 5 min

Spin 10,000 rpm 1 min

Take eluate and place back onto same column

Incubate 5 min, spin 10,000 rpm 1 min

Quantitate RNA.