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(RNA Later Method) October 09, 2000 [Use RNase-free eppendorf tubes & tips]2. Store sample (with RNA later) at 4oC for up to 6 hrs (RNA decay is observed after 6 hrs) (It is best if RNA is isolated immediately). 3. Pellet cells at 4oC using the Allegra 21R Beckman centrifuge and F0630 rotor (rotor pre-cooled and stored at 4oC), spin for 15-30 min at 10,000 rpm (longer times for larger volume harvests). Note: RNA Later prevents cells from pelletting well, use care when decanting supernatant. 5. Carefully pull off supernatant with glass pipet and resuspend pellet in 200 ul ice-cold lysis solution (lysozyme-containing TE as described in Qiagen RNeasy Kit). 6. Follow RNeasy protocol from Qiagen (If harvest O.D. was over 0.3 then split sample and use 2x Qiagen solutions and 2 columns). Note: You will over-load the columns if you don’t! 7. Collect final eluents from RNeasy preparation and combine duplicate samples into one 1.5 ml epp-tube and speed-vac sample (at 45 or 60oC) until it is almost completely dry. If it does happen to completely dry out that is OK, Resuspend RNA in 10-50 ul RNA free H2O. 8. Determine RNA concentration by measuring A260. 9. Storage of RNA samples: Add 2 volumes of 100% ethanol to RNA in water. Store RNA in ethanol at –80oC. 10. Mix well before removing aliquot. Remove required volume and precipitate, wash, then proceed as usual. Lysis Solution: |