<html xmlns:v="urn:schemas-microsoft-com:vml"
xmlns:o="urn:schemas-microsoft-com:office:office"
xmlns:w="urn:schemas-microsoft-com:office:word"
xmlns="http://www.w3.org/TR/REC-html40">
<head>
<meta name=Title content="Four μl PCR reaction-mix are analyzed using a 0">
<meta name=Keywords content="">
<meta http-equiv=Content-Type content="text/html; charset=macintosh">
<meta name=ProgId content=Word.Document>
<meta name=Generator content="Microsoft Word 11">
<meta name=Originator content="Microsoft Word 11">
<link rel=File-List href="Agarose%20electrophoresis_files/filelist.xml">
<title>Four μl PCR reaction-mix are analyzed using a 0</title>
<style>
</style>
</head>
<body bgcolor=white lang=EN-US link=blue vlink=purple style='tab-interval:36.0pt'>
|
Agarose
electrophoresis of DNA product<o:p></o:p>
- Analyse 4 μl PCR reaction-mix using
0.8% agarose gel electrophoresis and 5× RapidRun Loading Dye (1 μl, <a
href="http://www.usbweb.com/category.asp?cat=cre&id=77524">77524, usb</a>) to verify whether sufficient amount of DNA
was produced. <o:p></o:p>
- Agarose gel is prepared in 1× TBE
(Tris-borate-EDTA) buffer: <o:p></o:p>
- Prepare 10× TBE:<o:p></o:p>
- Tris base 108
g<o:p></o:p>
- Boric acid 55
g<o:p></o:p>
- EDTA (0.5M, pH 8.0) 40
ml<o:p></o:p>
- Make up to 1 L with autoclaved deionized
water.<o:p></o:p>
- Prepare <a href="file://localhost/Products.php">agarose</a> gel: <o:p></o:p>
- Agarose (<1000 BP) 1.6
g<o:p></o:p>
- 1× TBE 200
ml<o:p></o:p>
- Microwave at 1-min intervals until
completely dissolved.<o:p></o:p>
- Release the pressure inside the agarose
bottle after each 60-sec interval in a fume hood.<o:p></o:p>
- After cool down (~50 °C), add <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">ethidium bromide (EtBr)</a>; the stock is 10 μg/μl (1%
solution) 10
μl (0.05% final concentration)<o:p></o:p>
- Assemble <a href="file://localhost/Products.php">the gel mould</a>. In the fume hood, pour agarose up to about
half the height of the teeth of the gel comb. Take extreme care when handling ethidium bromide
(EtBr) and EtBr-containing gels.
Work in the hood.<o:p></o:p>
- Immerse gel in running buffer (1× TBE
buffer) in the <a
href="file://localhost/Products.php">running chamber</a>, place colored tape under lanes to help
visualization of the wells (if desired)<o:p></o:p>
- Load the gel wells with samples taking note
of the loading order. Make
sure the gel end with lanes is towards the black electrode (cathode). Run at 100V for 15–20 min. Visualize DNA under UV and take
a picture using Spencer Lab’s camera.<o:p></o:p>
<![if !supportEmptyParas]> <![endif]><o:p></o:p>
<a
href="http://openwetware.org/wiki/Bitan:todo">Back
to the To-Do List</a><o:p></o:p>
|
<![if !supportEmptyParas]> <![endif]><o:p></o:p>
</body>
</html>
