Bitan:Extraction of RNA from filters
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RNA extraction from the filters<o:p></o:p>
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RNA is extracted from the filters to obtain the sequences that bind to the protein. These sequences are amplified for the next SELEX cycle.<o:p></o:p>
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1) After <a href="http://openwetware.org/wiki/Bitan:Scintillation_counting_using_the_Triathler_bench-top_counter">scintillation counting</a>, remove the positive-control filter from the Eppendorf tube (<a href="http://openwetware.org/wiki/Bitan:Filter_binding_for_SELEX">from previous experiment</a>) and place into a clean, dry, 35×10-mm Petri dish. <o:p></o:p>
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2) Use a clean scalpel and a pair of tweezers to cut the membrane in small pieces.<o:p></o:p>
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3) Using the tweezers, transfer the cut pieces of the membrane into the same labeled Eppendorf tube from Step 1.<o:p></o:p>
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4) Add 400 μl elution buffer (7 M urea, 3 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 100 mM sodium citrate, pH 5.0) and incubate the tube at 95 °C for 10 min.<o:p></o:p>
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5) Centrifuge the tube at top speed, aspirate, and collect the extraction butter into a new labeled Eppendorf tube.<o:p></o:p>
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6) Measure the remaining radioactivity counts in the tube containing the membrane pieces by scintillation counting to assess the extraction efficiency.<o:p></o:p>
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7) Repeat the extraction process (steps 4–6) thrice. The efficiency after 3 extractions is usually ~95–96%.<o:p></o:p>
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8) In the tubes containing the RNA extracts, add 1 volume (400 μl) of citrate-saturated phenol (pH 4.7):chloroform:isoamyl alcohol (125:24:1). Mix by a vortex for ~1 minute and centrifuge at 16,000 g for 2 minutes.<o:p></o:p>
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9) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette. <o:p></o:p>
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10) Add 1 volume of chloroform:isoamyl alcohol (24:1), mix by a vortex for 1 minute and centrifuge at 16,000 g for 2 min. <o:p></o:p>
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11) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette. <o:p></o:p>
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12) To precipitate the RNA, add 0.1 volume of 3 M sodium acetate (pH 5.2), 3–4 μl glycogen (10 μg/μl) as the coprecipitant, and 1 volume equivalent of propan-2-ol. Mix and place in a −20 °C freezer overnight. <o:p></o:p>
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13) Spin at top speed, preferably in a microcentrifuge at 4 °C, for 20–30 minutes to precipitate the RNA from step 12.<o:p></o:p>
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14) After centrifugation, aspirate the supernate carefully without dislodging the coprecipitant phase barely visible in the tube. <o:p></o:p>
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15) Wash the RNA pellet with 0.5 ml of 70% ethanol, centrifuge for 5 min a top speed and discard ethanol by aspiration without dislodging the coprecipitant phase barely visible in the tube.<o:p></o:p>
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16) Dissolve the RNA pellet in 50 μl STE buffer and proceed to <a href="http://openwetware.org/wiki/Bitan:In-vitro_transcription%2C_labeling_and_G-50_purification_of_RNA">G-50 purification</a>.<o:p></o:p>
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<a href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a>.<o:p></o:p>
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