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.
