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: 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 RNA 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. |
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