Bitan:Agarose gel electrophoresis



       Four &#956;l PCR reaction-mix are analyzed using a 0



   Agarose electrophoresis of DNA product <ol style='margin-top:0cm' start=1 type=1> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Analyse 4 &#956;l PCR reaction-mix using 0.8% agarose gel electrophoresis and 5&times; <span style='font-family:Calibri;mso-fareast-language:JA'> <span style='font-family:Calibri'>RapidRun Loading Dye (1 &#956;l, <span      style='font-family:Times'><a       href="http://www.usbweb.com/category.asp?cat=cre&amp;id=77524"><span       style='font-family:Calibri;color:windowtext'>77524, usb </a> <span       style='font-family:Calibri'>) to verify whether sufficient amount of DNA was produced. </o:p> </li>

<li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Agarose gel is prepared in 1&times; TBE (Tris-borate-EDTA) buffer: </o:p> </li> <ol style='margin-top:0cm' start=1 type=a> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 108.0pt'><span style='font-family:Calibri'>Prepare 10&times; TBE:</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 108.0pt'><span style='font-family:Calibri'>Tris base                                108 g</o:p> </li>

<li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 108.0pt'><span style='font-family:Calibri'>Boric acid                               55 g</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 108.0pt'><span style='font-family:Calibri'>EDTA (0.5M, pH 8.0)<span style='mso-tab-count: 2'>              40        ml</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 108.0pt'><span style='font-family:Calibri'>Make up to 1 L with autoclaved deionized water.</o:p> </li> </ol> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Prepare <span style='font-family: Times'><a href="file://localhost/Products.php"><span style='font-family: Calibri;color:windowtext'>agarose </a> <span style='font-family:Calibri'> gel: </o:p> </li>

<ol style='margin-top:0cm' start=1 type=a> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span style='font-family:Calibri'>Agarose (&lt;1000 BP)<span style='mso-tab-count:2'>               1.6 g</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span style='font-family:Calibri'>1&times; TBE<span style='mso-tab-count: 3'>                                    200        ml</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span style='font-family:Calibri'>Microwave at 1-min intervals until completely dissolved.</o:p> </li>

<li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span style='font-family:Calibri'>Release the pressure inside the agarose bottle after each 60-sec interval in a fume hood.</o:p> </li> <li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span style='font-family:Calibri'>After cool down (~50 °C), add <span style='font-family:Times'><a href="http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&amp;productId=734529&amp;catalogId=29104&amp;matchedCatNo=BP130210&amp;pos=1&amp;catCode=RE_SC&amp;endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&amp;fromCat=yes&amp;keepSessionSearchOutPut=true&amp;fromSearch=Y&amp;searchKey=bromide%7C%7Cethidium&amp;highlightProductsItemsFlag=Y"><span style='font-family:Calibri;color:windowtext'>ethidium bromide (EtBr) </a> <span style='font-family:Calibri'>; the stock is 10 &#956;g/&#956;l (1%       solution)                            10 &#956;l (0.05% final concentration)</o:p> </li> </ol> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Assemble <span style='font-family: Times'><a href="file://localhost/Products.php"><span style='font-family: Calibri;color:windowtext'>the gel mould </a> <span style='font-family:Calibri'>. In the fume hood, pour agarose up to about half the height of the teeth of the gel comb.<span style="mso-spacerun:      yes">  Take extreme care when handling ethidium bromide (EtBr) and EtBr-containing gels.

Work in the hood.</o:p> </li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Immerse gel in running buffer (1&times; TBE      buffer) in the <a href="file://localhost/Products.php"><span style='font-family:Calibri; color:windowtext'>running chamber </a> <span style='font-family:Calibri'>, place colored tape under lanes to help visualization of the wells (if desired)</o:p> </li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list 36.0pt left 144.0pt'><span style='font-family:Calibri'>Load the gel wells with samples taking note of the loading order. Make sure the gel end with lanes <span style='font-family:Calibri; mso-fareast-language:JA'>is towards the black electrode (cathode). <span style='font-family:Calibri'>Run at 100V for 15&#8211;20 min.<span style="mso-spacerun: yes"> Visualize DNA under UV and take a picture using Spencer Lab’s camera.</o:p> </li>

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