Bitan:Agarose gel electrophoresis
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<p class=MsoNormal align=center style='margin-left:18.0pt;text-align:center;
tab-stops:144.0pt'><span style='font-size:14.0pt;font-family:Calibri'><b>Agarose
electrophoresis of DNA product<o:p></o:p></b></span></p>
<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 μl PCR reaction-mix using
0.8% agarose gel electrophoresis and 5×</span><span
style='font-family:Calibri;mso-fareast-language:JA'> </span><span
style='font-family:Calibri'>RapidRun Loading Dye (1 μl, </span><span
style='font-family:Times'><a
href="http://www.usbweb.com/category.asp?cat=cre&id=77524"><span
style='font-family:Calibri;color:windowtext'>77524, usb</span></a></span><span
style='font-family:Calibri'>) to verify whether sufficient amount of DNA
was produced.<span style="mso-spacerun: yes"> </span><o:p></o:p></span></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× TBE
(Tris-borate-EDTA) buffer: <o:p></o:p></span></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× TBE:<o:p></o:p></span></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<span style='mso-tab-count:3'> </span>108
g<o:p></o:p></span></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<span style='mso-tab-count:3'> </span>55
g<o:p></o:p></span></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'> </span>40
ml<o:p></o:p></span></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></o:p></span></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><span style='font-family:
Times'><a href="file://localhost/Products.php"><span style='font-family:
Calibri;color:windowtext'>agarose</span></a></span><span
style='font-family:Calibri'> gel: <o:p></o:p></span></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 (<1000 BP)<span
style='mso-tab-count:2'> </span>1.6
g<o:p></o:p></span></li>
<li class=MsoNormal style='mso-list:l0 level2 lfo1;tab-stops:list 72.0pt left 144.0pt'><span
style='font-family:Calibri'>1× TBE<span style='mso-tab-count:
3'> </span>200
ml<o:p></o:p></span></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></o:p></span></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></o:p></span></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><span
style='font-family:Times'><a
href="http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&productId=734529&catalogId=29104&matchedCatNo=BP130210&pos=1&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=bromide%7C%7Cethidium&highlightProductsItemsFlag=Y"><span
style='font-family:Calibri;color:windowtext'>ethidium bromide (EtBr)</span></a></span><span
style='font-family:Calibri'>; the stock is 10 μg/μl (1%
solution)<span style='mso-tab-count:3'> </span>10
μl (0.05% final concentration)<o:p></o:p></span></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><span style='font-family:
Times'><a href="file://localhost/Products.php"><span style='font-family:
Calibri;color:windowtext'>the gel mould</span></a></span><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"> </span>Take extreme care when handling ethidium bromide
(EtBr) and EtBr-containing gels.<span style="mso-spacerun: yes">
</span>Work in the hood.<o:p></o:p></span></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× TBE
buffer) in the </span><span style='font-family:Times'><a
href="file://localhost/Products.php"><span style='font-family:Calibri;
color:windowtext'>running chamber</span></a></span><span
style='font-family:Calibri'>, place colored tape under lanes to help
visualization of the wells (if desired)<o:p></o:p></span></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.<span style="mso-spacerun: yes"> </span>Make
sure the gel end with lanes </span><span style='font-family:Calibri;
mso-fareast-language:JA'>is towards the black electrode (cathode). </span><span
style='font-family:Calibri'>Run at 100V for 15–20 min.<span
style="mso-spacerun: yes"> </span>Visualize DNA under UV and take
a picture using Spencer Lab’s camera.<o:p></o:p></span></li>
</ol> <p class=MsoNormal align=center style='text-align:center;tab-stops:144.0pt'><span style='font-size:14.0pt;font-family:Calibri'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal align=center style='text-align:center;tab-stops:144.0pt'><span style='font-size:14.0pt;font-family:Calibri'><a href="http://openwetware.org/wiki/Bitan:todo"><span style='color:windowtext'>Back to the To-Do List</span></a></span><span style='font-size:14.0pt;font-family: Times'><o:p></o:p></span></p> </td> </tr>
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