Pat Brown's Lab ,Department of Biochemistry and the Howard Hughes Medical Institute
http://cmgm.stanford.edu/pbrown/protocols/short_paxgene_protocol.html
Short PAXgene Protocol:
- spin in step 6 increased to 20 min; no Dnase treatment; optional wash after step 12 REQUIRED
1. Centrifuge the PAXgene Blood RNA Tube for 10 min at 3000–5000 x g using a swing-out rotor.
Note: Ensure that the blood sample has been incubated in the PAXgene Blood RNA Tube for a minimum of 2 h at room temperature, in order to achieve complete lysis.
Preheat a water bath or heat block (for microcentrifuge tubes) to 55°C, during first spin.
We spin at 3750 rpm in a standard table top centrifuge.
2. Remove the supernatant by decanting or pipetting. Add 5 ml RNase-free water to the pellet, and close the tube using a fresh secondary Hemogard closure.
Gently decant the supernatant, and blot on a paper towel.
3. Thoroughly resuspend the pellet by vortexing, and centrifuge for 10 min at 3000–5000 x g. Remove and discard the entire supernatant.
Small debris remaining in the supernatant after vortexing but before centrifugation will not affect the procedure.
Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate which will affect the conditions for binding RNA to the PAXgene membrane.
Pellet does NOT wash to clear/white color; will remain brown.
4. Thoroughly resuspend the pellet in 360 µl Buffer BR1 by vortexing.
5. Pipet the sample into a 1.5 ml or 2 ml microcentrifuge tube (not supplied). Add 300 µl Buffer BR2 and 40 µl Proteinase K. Mix by vortexing, and incubate for 10 min at 55°C using a shaker–incubator, heating block, or water bath.
If a heating block or water bath is used, vortex each sample once during the incubation. Do not allow the temperature of the sample to decrease during vortexing.
Note: Do not mix Buffer BR2 and Proteinase K together before adding them to the sample.
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