| Protocol from Invitrogen technical support
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)
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For 1ml: 200 mM Tris, pH 8.4 200 ul 1M Tris, pH 8.4 500 ul 1M KCl 20 ul 1 M MgCl2 280 ul DEPC water
- 25 mM EDTA
- RNeasy 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 buffer DEPC water to 10 ul
Example of a scaled up version for a 40 ul reaction100 ug Total RNA4 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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