Bitan:Filter binding for SELEX



       RNA&#8211;protein incubation and filter binding:



RNA&#8211;protein incubation and filter binding for SELEX:

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<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>In this step, the RNA and protein are first incubated in solution and then the RNA sequences that bind to the protein are separated from the non-binders by filter binding and washing. As SELEX cycles progress, filter binding will give an indication of protein&#8211;RNA binding enrichment.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>1) Dissolve the protein in 8 &#956;l 60 mM NaOH and add 36 &#956;l nuclease-free deionized water.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>2) Sonicate the mixture for 1 min. Add 36 &#956;l 2&times; RNA binding buffer (20 mM Tris, 300 mM NaCl, 10 mM MgCl2, pH 7.5).</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>3) Incubate the RNA at 90 °C for 10 min for denaturation and then incubate at room temperature for 10 min for slow RNA renaturation.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>4) Mix the appropriate amount of RNA with 20 &#956;l 10&times; RNA binding buffer and make up the volume to 200 &#956;l by adding nuclease-free water. This is the negative control; label the tube negative control.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>5) Mix 20 &#956;l protein and the desired amount of RNA with 20 &#956;l 10&times; RNA binding buffer and make up the volume to 200 &#956;l by adding nuclease-free water. This is the positive control.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>6) Mix and incubate the tubes for 30 min at room temperature. Meanwhile prepare the filters and the filter-binding setup for the next step.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>7) Attach a 125-ml side-arm flask to a vacuum inlet. Place a pre-cleaned, porous glass support for the filter on the side-arm flask.</o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>8) Equilibrate three filter membranes in 2&#8211;3 ml of 1&times; RNA binding buffer in a 35&times;10-mm Petri dish for 10&#8211;15 min. The first filter will be used for adjusting the vacuum suction, the second will be used for RNA-alone, negative-control filter binding, and the third will be used for filter binding of the RNA&#8211;protein mixture, the positive control.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>9) After 30 min, centrifuge the negative- and positive-control for 5 min. <o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>10) Turn on the vacuum, and place the first membrane on the porous glass. Using a micropipette, drip 0.5 ml of RNA binding buffer onto the membrane and adjust the vacuum to allow slow flow of each buffer drop through the membrane. <o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>11) Place the second membrane onto the porous glass and using the same flow rate, apply the negative-control onto the membrane. <o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>12) Apply 4&times;0.5-ml aliquots of 1&times; RNA binding buffer to wash the membrane and discard the flow through. Note that if pre-clearing is desired, the flow-through is kept for RNA extraction. Pre-clearing removes RNA sequences that bind to the filter.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>13) Remove the negative-control membrane and place into a correspondingly labeled 1.6-ml Eppendorf for scintillation counting.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>14) Replace the porous glass with a pre-cleaned second porous glass support.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>15) Place the third disk onto the porous glass and apply the positive-control. Wash the filter as in step 12 and discard the flow-through. The number of washes can be increased in later SELEX cycles to increase the stringency of SELEX conditions.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>16) Remove the positive-control filter disk and place into a correspondingly labeled 1.6-ml Eppendorf for scintillation counting.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>17) Perform <a href="http://openwetware.org/wiki/Bitan:Scintillation_counting_using_the_Triathler_bench-top_counter"><span style='font-family:Century'>scintillation counting </a> <span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'> as before and note down the counts for the respective filters.<o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:Century;mso-fareast-language:KO'>18) Calculate the level of the filter-bound radioactivity compared to the total amount of radioactivity applied to the membranes (% binding). This will give an indication of binding enrichment as SELEX progresses.<o:p></o:p>

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