Team:KIT-Kyoto/Notebook/Protocol

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Latest revision as of 01:40, 28 September 2013

Protocol

Protocol

Miniprep

Solution I (50 mM Tris-HCl and 10 mM ED TA, and 50 µg/mL RNase A, pH 8.0 (25˚C))

Solution II (0.2 M NaOH and 1 % SDS)

Solution III (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)

Solution IV (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))

Solution V (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))

・Pick up a single colony from plate and cultivate it overnight in 3mL LB medium containing appropriate antibiotic at 37˚C.

・Transfer the culture into a 1.5mL tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).

・Add 250 µL of SolutionI to the pellet and mix well with vortex.

・Add 250 µL of SolutionII and mix well by turning the tube upside down several times.

・Add 350 µL of SolutionIII and by turning the tube upside down several times.

・Centrifuge at 13,000 rpm for 5 minutes.

・Transfer the supernatant (approx. 850µL) to a mini prep column.

・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

・Add 500 µL of SolutionIV.

・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

・Add 700 µL of SolutionV.

・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).

・Set the column on a new 1.5mL tube and add 100 µL of nuclease-free water.

・Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.

Rapid check of the insert by colony cracking

・Add 45 µL of cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.

・Suspend a small quantity of the colony in a tube using a sterilized toothpick.

・Incubate the tube at 65°C for 10 minutes.

・Add a drop of 10x Loading Buffer.

・Add equal volume of phenol/chloroform.

・Mix with vortex.

・Centrifuge at 13,000rpm for 3 minutes.

・Apply the supernatant to 1% agarose gel electrophoresis.

・Stain the gel in ethidium bromide solution for 10 minutes.

・Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.

DNA purification and precipitation

・Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.

・Mix DNA solution with equal volume of phenol/chloroform by vortex.

・Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.

・Add equal volume of 2-propanol, and mix by turning the tube upside down.

・Centrifuge at 14,500 rpm for 10 min at 4˚C.

・Carefully decant the supernatant.

・Add adequate volume of 70 % ethanol.

・Centrifuge at 14,500 rpm for 5 min at 4˚C.

・Carefully decant the supernatant.

・Dry in the desiccator under vacuum for 5 min.

・You can get dried DNA pellet.

PCR

・Adjust the concentration of each primer to 100 pmol/µL with sterilized water.

・Mix 10µL of forward and reverse primer solutions with 80 µL H₂O in a new tube (final primer concentration is 10 pmol/µL).

・Use 1 µL of primer mix for PCR.

Recipe for PRC is as follows:

Buffer	50 µL
dNTP	20 µL
Primer mix	1 µL
DNA sample	0.5 µL
KOD-FX	2 µL
H₂O	26.5 µL
total	100 µL

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

12.5% separation gel (recipe for a sheet of gel)

Mili Q water	2.59 mL
acrylamide solution(30%)	3.33 mL
0.5M Tris(pH8.8)	2 mL
10%SDS	80 µL
10%APS	27 µL
TEMED	4 µL

・Apply the acrylamide solution mix to the PAGE glass plate.

・Deposit H₂O carefully on the top of the acrylamide solution mix.

・Wait for 10 min for polymerization.

Stacking gel

Mili Q water	2.89 mL
acrylamide	0.79 mL
0.5M Tris(pH6.8)	1.25 mL
10%SDS	50 µL
10%APS	17 µL
TEMED	5 µL

・After the polymerization of the separation gel, remove the H₂O of the top layer.

・Apply stacking gel mix on the running gel and put the comb to make wells.

・After the stacking gel has fully polymerized, remove the comb and rinse the top of the gel with H₂O and then remove H₂O.

Sample preparation

・Collect bacterial cells.

・Add 450µL of H₂O and 50 µL of FastBreak Cell Lysis Reagent (Promega,Madison,Wisconsin,USA).

・Mix them for 15minutes with shaker.

・Add 100µL of 6xSDS-Sample buffer and mix with vortex.

・Heat samples in a heating block at 99°C for 5 minutes.

Running samples

・Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%)

・Apply the samples and markers.

・Operate at constant current (25mA) for 65 minutes per sheet of gel.

Staining

・Place the gel in stacking solution(50%ethanol,10%acetic acid) and shake gently it for 5 minutes.

・Remove the stacking solution and add 100mL of staining solution (0.25% CBB R250、5%methanol、7.5%acetic acid).

・Warm the gel for 1 minute in a microwave (500W).

・Remove the staining solution and rinse the gel with water.

Isolation and purification of DNA bands

・Clip DNA band from agarose gel and transfer it into a 1.5 mL tube.

・Add H₂O. Melt the gel at 65˚C in a heating block.

・Add equal volume of phenol. Mix well with vortex.

・Centrifuge at 13,000 rpm for 5 min.

・Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.

・Perform the same method of DNA precipitation using 2-propanol.

Blue gel

1xTAE	100mL
Agarose(Nacalai tesque)	1.0g
Gel indicator(Biodynamics laboratory)	200 µL

Ligation

・Dissolve the purified and dried insert DNA in 5µL of H₂O.

・Dissolve the purified and dried vector DNA in 5µL of H₂O.

・Mix the two solutions.

・Add 10µL of Ligation Mix (Wako) to it.

・Incubate for 15 minutes at room temperature.

Transformation

・Add the ligation solution to competent cells (in a clean bench).

・Place tubes on ice for 20 minutes.

・Heat shock treatment at 42°C for 35 seconds.

・Place tubes on ice for 2 minutes.

・Add 1mL of LB medium into the tube (in a clean bench).

・Incubate the samples for 20 minutes at 37°C.

・Centrifuge at 7,000rpm for 3 minutes.

・Discard the most of supernatant, and spread the remaining cells and LD medium in the tube on the plates (in a clean bench).

・Incubate plates overnight at 37°C.

@@ Line 4: / Line 4: @@
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 <head>
-<style>
-#"doc-right{
-  padding-left : 20px;
-}
-</style>
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-</html>
+<body>
 <div class="document">
    <div id="doc-left">
-{{Template:KIT-Kyoto/menu_note}}
+  <ul class="nav metro-nav-list">
+   <li class="nav-header">Protocol</li>
+<ul class="nav">
+         <li><a href="#Miniprep">Miniprep</a></li>
+         <li><a href="#Rapid check of the insert by colony cracking">Rapid check of the insert by colony cracking</a></li>
+         <li><a href="#DNA purification and precipitation">DNA purification and precipitation</a></li>
+         <li><a href="#PCR">PCR</a></li>
+         <li><a href="#SDS polyacrylamide gel electrophoresis (SDS-PAGE)">SDS-PAGE</a></li>
+         <li><a href="#Isolation and purification of DNA bands">Isolation and purification of DNA bands</a></li>
+         <li><a href="#Ligation">Ligation</a></li>
+         <li><a href="#Transformation">Transformation</a></li>
+</ul>
+  </ul>
    </div>
 <div id="doc-right" class="metro">
+<div id="Protocol" style="margin-top:-70px; padding-top:70px;">
+<img src="https://static.igem.org/mediawiki/2013/3/32/Protocol_kit.JPG" width="900">
 <h2>Protocol</h2>
+<div id="Miniprep" style="margin-top:-70px; padding-top:70px;">
+<p><b><font size="5">Miniprep</font></b></p>
+<p>Solution I (50 mM Tris-HCl and 10 mM ED TA, and 50 µg/mL RNase A, pH 8.0 (25˚C))</p>
+<p>Solution II (0.2 M NaOH and 1 % SDS)</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Miniprep</B></FONT></SPAN></FONT></P>
+<p>Solution III (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)</p>
-<P STYLE="margin-bottom: 0cm">
+<p>Solution IV (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))</p>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+<p>Solution V (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))</p>
-&#8544; (50 mM Tris-HCl and 10 mM EDTA, and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">g/mL
+<br>
-RNase A, pH 8.0 (25&#730;C))</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+<p>・Pick up a single colony from plate and cultivate it overnight in 3mL LB medium containing appropriate antibiotic at 37˚C.</p>
-&#8545; (0.2 M NaOH and 1 % SDS)</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+<p>・Transfer the culture into a 1.5mL tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).</p>
-&#8546; (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
+<p>・Add 250 µL of SolutionI to the pellet and mix well with vortex.</p>
-&#8547; (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH
-.6 (25&#730;C))</FONT></SPAN></FONT></P>
+<p>・Add 250 µL of SolutionII and mix well by turning the tube upside down several times.</p>
+<p>・Add 350 µL of SolutionIII and by turning the tube upside down several times.</p>
+<p>・Centrifuge at 13,000 rpm for 5 minutes.</p>
+<p>・Transfer the supernatant (approx. 850µL) to a mini prep column.</p>
+<p>・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.</p>
+<p>・Add 500 µL of SolutionIV.</p>
+<p>・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.</p>
+<p>・Add 700 µL of SolutionV.</p>
+<p>・Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).</p>
+<p>・Set the column on a new 1.5mL tube and add 100 µL of nuclease-free water.</p>
+<p>・Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
-&#8548; (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25&#730;C))</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt"></FONT></FONT></FONT></P>
 <br>
+<div id="Rapid check of the insert by colony cracking" style="margin-top:-70px; padding-top:70px;">
+<p><b><font size="5">Rapid check of the insert by colony cracking</font></b></p>
+<p>・Add 45 µL of cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube. </p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Pick
+<p>・Suspend a small quantity of the colony in a tube using a sterilized toothpick.</p>
-up a single colony from plate and cultivate it overnight in 3ml LB
-medium containing appropriate antibiotic at 37&#730;C&#730;.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Incubate the tube at 65°C for 10 minutes.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
+<p>・Add a drop of 10x Loading Buffer.</p>
-the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute,
-and discard the supernatant (2 times).</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Add equal volume of phenol/chloroform.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p>・Mix with vortex.</p>
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-of Solution&#8544; to the pellet and mix well with vortex.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Centrifuge at 13,000rpm for 3 minutes.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p>・Apply the supernatant to 1% agarose gel electrophoresis.</p>
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-of Solution&#8545; and mix well by turning the tube upside down several
-times.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Stain the gel in ethidium bromide solution for 10 minutes.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p>・Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.</p>
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-of Solution&#8546; and by turning the tube upside down several times.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<br>
+<div id="DNA purification and precipitation" style="margin-top:-70px; padding-top:70px;">
+<p><b><font size="5">DNA purification and precipitation</font></b></p>
+<p>・Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<p>・Mix DNA solution with equal volume of phenol/chloroform by vortex.</p>
-at 13,000 rpm for 5 minutes.</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
+<p>・Add equal volume of 2-propanol, and mix by turning the tube upside down.</p>
-the supernatant (approx. 850</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L)
-to a mini prep column.</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・Centrifuge at 14,500 rpm for 10 min at 4˚C.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<p>・Carefully decant the supernatant.</p>
-at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・Add adequate volume of 70 % ethanol.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p>・Centrifuge at 14,500 rpm for 5 min at 4˚C.</p>
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-of Solution&#8547;.</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・Carefully decant the supernatant.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<p>・Dry in the desiccator under vacuum for 5 min.</p>
-at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・You can get dried DNA pellet.</p>
+<br>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<div id="PCR" style="margin-top:-70px; padding-top:70px;">
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+<p><b><font size="5">PCR</font></b></p>
-of Solution&#8548;.</FONT></SPAN></FONT></P>
+<p>・Adjust the concentration of each primer to 100 pmol/µL with sterilized water.</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<p>・Mix 10µL of forward and reverse primer solutions with 80 µL H<SUB>2</SUB>O in a new tube (final primer concentration is 10 pmol/µL).</p>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<p>・Use 1 µL of primer mix for PCR.</p>
-at 13,000 rpm for 1 minute and discard the flow-through (2 times).</FONT></SPAN></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<br>
+Recipe for PRC is as follows:
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
+<Table Border Cellspacing="0">
-the column on a new 1.5ml tube and add 100 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
+<Tr><Td>　Buffer　</Td><Td>　50 &micro;L　</Td></Tr>
-of nuclease-free water.</FONT></SPAN></FONT></P>
+<Tr><Td>　dNTP　</Td><Td>　20 &micro;L　</Td></Tr>
+<Tr><Td>　Primer mix　</Td><Td>　1 &micro;L　</Td></Tr>
+<Tr><Td>　DNA sample　</Td><Td>　0.5 &micro;L　</Td></Tr>
+<Tr><Td>　KOD-FX　</Td><Td>　2 &micro;L　</Td></Tr>
+<Tr><Td>　H<SUB>2</SUB>O　</Td><Td>　26.5 &micro;L　</Td></Tr>
+<Tr><Td>　total　</Td><Td>　100 &micro;L　</Td></Tr>
+</Table>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
+<br>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<div id="SDS polyacrylamide gel electrophoresis (SDS-PAGE)" style="margin-top:-70px; padding-top:70px;">
-at 13,000 rpm for 1 minute and collect plasmid DNA.</FONT></SPAN></FONT></P>
+<p><b><font size="5">SDS polyacrylamide gel electrophoresis (SDS-PAGE)</font></b></p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">	</SPAN></FONT></P>
+<p><b><font size="3">12.5% separation gel (recipe for a sheet of gel)</font></b></p>
+<Table Border Cellspacing="0">
+<Tr><Td>　Mili Q water　</Td><Td>　2.59 mL　</Td></Tr>
+<Tr><Td>　acrylamide solution(30%)　</Td><Td>　3.33 mL　</Td></Tr>
+<Tr><Td>　0.5M Tris(pH8.8)　</Td><Td>　2 mL　</Td></Tr>
+<Tr><Td>　10%SDS　</Td><Td>　80 &micro;L　</Td></Tr>
+<Tr><Td>　10%APS　</Td><Td>　27 &micro;L　</Td></Tr>
+<Tr><Td>　TEMED　</Td><Td>　4 &micro;L　</Td></Tr>
+</Table>
+<br>
+<p>・Apply the acrylamide solution mix to the PAGE glass plate.</p>
-<BR>
+<p>・Deposit H<SUB>2</SUB>O carefully on the top of the acrylamide solution mix.</p>
-<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Rapid
+<p>・Wait for 10 min for polymerization.</p>
-check of the insert by colony cracking</B></FONT></SPAN></FONT></P>
+<br>
-<P STYLE="margin-bottom: 0cm">
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p><b><font size="3">Stacking gel </font></b></p>
-</FONT>&micro;L<FONT SIZE=2 STYLE="font-size: 11pt"> of cracking
+<Table Border Cellspacing="0">
-solution into each tube.   </FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">Cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6)</FONT></FONT></FONT></P>
+<Tr><Td>　Mili Q water　</Td><Td>　2.89 mL　</Td></Tr>
+<Tr><Td>　acrylamide　</Td><Td>　0.79 mL　</Td></Tr>
+<Tr><Td>　0.5M Tris(pH6.8)　</Td><Td>　1.25 mL　</Td></Tr>
+<Tr><Td>　10%SDS　</Td><Td>　50 &micro;L　</Td></Tr>
+<Tr><Td>　10%APS　</Td><Td>　17 &micro;L　</Td></Tr>
+<Tr><Td>　TEMED　</Td><Td>　5 &micro;L　</Td></Tr>
+</Table>
+<br>
+<p>・After the polymerization of the separation gel, remove the H<SUB>2</SUB>O of the top layer.</p>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Apply stacking gel mix on the running gel and put the comb to make wells.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Suspend
+<p>・After the stacking gel has fully polymerized, remove the comb and rinse the top of the gel with H<SUB>2</SUB>O and then remove H<SUB>2</SUB>O.</p>
-a small quantity of the colony in a tube using a sterilized
-toothpick.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<br>
+<p><b><font size="3">Sample preparation </font></b></p>
+<p>・Collect bacterial cells.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Incubate
+<p>・Add 450µL of H<SUB>2</SUB>O and 50 µL of FastBreak Cell Lysis Reagent (Promega,Madison,Wisconsin,USA).</p>
-the tube at 65&#176;C for 10 minutes.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Mix them for 15minutes with shaker.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<p>・Add 100µL of 6xSDS-Sample buffer and mix with vortex.</p>
-a drop of 10x Loading Buffer.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Heat samples in a heating block at 99°C for 5 minutes.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
+<br>
-equal volume of phenol/chloroform.</FONT></SPAN></FONT></P>
+<p><b><font size="3">Running samples </font></b></p>
+<p>・Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%)  </p>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Apply the samples and markers.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
+<p>・Operate at constant current (25mA) for 65 minutes per sheet of gel.</p>
-with vortex.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<br>
+<p><b><font size="3">Staining </font></b></p>
+<p>・Place the gel in stacking solution(50%ethanol,10%acetic acid) and shake gently it for 5 minutes.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
+<p>・Remove the stacking solution and add 100mL of staining solution (0.25% CBB R250、5%methanol、7.5%acetic acid).</p>
-at 13,000rpm for 3 minutes.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Warm the gel for 1 minute in a microwave (500W).</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
+<p>・Remove the staining solution and rinse the gel with water.</p>
-the supernatant to 1% agarose gel electrophoresis.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<br>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Stain
+<div id="Isolation and purification of DNA bands" style="margin-top:-70px; padding-top:70px;">
-the gel in ethidium bromide solution for 10 minutes.</FONT></SPAN></FONT></P>
+<p><b><font size="5">Isolation and purification of DNA bands</font></b></p>
+<p>・Clip DNA band from agarose gel and transfer it into a 1.5 mL tube.</p>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
+<p>・Add H<SUB>2</SUB>O. Melt the gel at 65˚C in a heating block.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Plasmid
+<p>・Add equal volume of phenol. Mix well with vortex.</p>
-DNA can be detected by UV-illuminator as a band between genomic DNA
-band and low molecular size RNAs.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
+<p>・Centrifuge at 13,000 rpm for 5 min.</p>
-</P>
-<UL>
+<p>・Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.</p>
-	<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B>
-	</B></FONT><FONT FACE=""><SPAN LANG="en-US"><B>DNA purification and
-	precipitation</B></SPAN></FONT></P>
-</UL>
-<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
+<p>・Perform the same method of DNA precipitation using 2-propanol.</p>
-</P>
-<OL>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
-&#181;L of sterilized water and 50 &#181;L of 3 M sodium
-	acetate with DNA sample.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
-	DNA solution with equal volume of phenol/chloroform by vortex.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
-	at 13,000 rpm for 5 min, then transfer the supernatant into a new
-	tube.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
-	equal volume of 2-propanol, and mix by turning the tube upside down.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
-	at 14,500 rpm for 10 min at 4&#730;C.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
-	decant the supernatant.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
-	adequate volume of 70 % ethanol.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
-	at 14,500 rpm for 5 min at 4&#730;C.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
-	decant the supernatant.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Dry
-	in the desiccator under vacuum for 5 min.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">You
-	can get dried DNA pellet.</SPAN></FONT></P>
-</OL>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"></FONT></FONT></P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm; page-break-before: always"><BR>
-</P>
-<UL>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>PCR</B></SPAN></FONT></P>
-</UL>
-<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
-</P>
-<OL>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Adjust
-	the concentration of each primer to 100 pmol/&micro;L with
-	sterilized water.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
-&micro;L of forward and reverse primer solutions with 80 &micro;L
-	H<SUB>2</SUB>O in a new tube (final primer concentration is 10
-	pmol/&micro;L).</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Use
-&micro;L of primer mix for PCR.</SPAN></FONT></P>
-</OL>
-<P STYLE="margin-left: 1.48cm; margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Recipe
-for PRC is as follows:</SPAN></FONT></P>
-<TABLE WIDTH=206 BORDER=1 BORDERCOLOR="#00000a" CELLPADDING=7 CELLSPACING=0>
-	<COL WIDTH=106>
-	<COL WIDTH=70>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">Buffer</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P><FONT FACE=""><SPAN LANG="en-US">50 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">dNTP</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P><FONT FACE=""><SPAN LANG="en-US">20 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">Primer mix</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P> <FONT FACE=""><SPAN LANG="en-US">1 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">DNA sample</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P><FONT FACE=""><SPAN LANG="en-US">0.5 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P> <FONT FACE=""><SPAN LANG="en-US">2 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">H<SUB>2</SUB>O</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P><FONT FACE=""><SPAN LANG="en-US">26.5 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-	<TR VALIGN=TOP>
-		<TD WIDTH=106>
-			<P><FONT FACE=""><SPAN LANG="en-US">total</SPAN></FONT></P>
-		</TD>
-		<TD WIDTH=70>
-			<P><FONT FACE=""><SPAN LANG="en-US">100 &micro;L</SPAN></FONT></P>
-		</TD>
-	</TR>
-</TABLE>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT><FONT FACE=""></FONT><FONT FACE=""><SPAN LANG="en-US">(</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Toyobo</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS
-polyacrylamide gel electrophoresis (SDS-PAGE)</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>12.5%
-separation gel (recipe for a sheet of gel)</B></FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
-Q water			2.59 ml </FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">acrylamide
-solution(</FONT><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">30
-</FONT></FONT><FONT SIZE=2 STYLE="font-size: 11pt">%)	3.33 ml</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
-Tris (pH8.8)		2 ml </FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%SDS			80
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-</FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS			27
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-</FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED			4
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-</FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
-the acrylamide solution mix to the PAGE glass plate.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Deposit
-H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
-carefully on the top of the acrylamide solution mix.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Wait
-for 10 min for polymerization.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Stacking
-gel  </B></FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
-Q water 	2.89 ml</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Acrylamide
-.79 ml</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
-Tris (pH 6.8) 	1.25 ml</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%
-SDS 	50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED
-</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
-the polymerization of the separation gel, remove the H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
-of the top layer.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
-stacking gel mix on the running gel and put the comb to make wells.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
-the stacking gel has fully polymerized, remove the comb and rinse the
-top of the gel with H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
-and then remove H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Sample
-preparation</B></FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Collect
-bacterial cells.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
-ul of H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
-and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
-of FastBreak Cell Lysis Reagent (</FONT><FONT SIZE=2 STYLE="font-size: 11pt">Madison,
-Wisconsin, USA</FONT><FONT SIZE=2 STYLE="font-size: 11pt"> Promega).</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
-them for 15minutes with shaker.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
-ul of 6</FONT></SPAN></FONT><FONT FACE=""><FONT SIZE=2 STYLE="font-size: 11pt">ｘ</FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS-Sample
-buffer and mix with vortex.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Heat
-samples in a heating block at 99&#176;C for 5 minutes.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Running
-samples</B></FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
-the gel on the electrophoresis apparatus and apply SDS running buffer
-(</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">Tris 25mM,Glysine 191mM,SDS0.1%</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
-</FONT></SPAN></FONT>
-</P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
-the samples and markers.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Operate
-at constant current (25mA) for 65 minutes per sheet of gel.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Staining</B></FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Place
-the gel in stacking solution( </FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">50％ethanol,10％acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
-and shake gently it for 5 minutes.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
-the stacking solution and add 100ml of staining solution (</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">0.25% CBB R250、5%methanol、7.5%acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">).</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Warm
-the gel for 1 minute with a microwave (500W).</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm">&darr;</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
-the staining solution and rinse the gel with water.</FONT></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<UL>
-	<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B></B></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>Isolation
-	and purification of DNA bands</B></SPAN></FONT></P>
-</UL>
-<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
-</P>
-<OL>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Clip
-	DNA band from agarose gel and transfer it into a 1.5 ml tube.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
-	H<SUB>2</SUB>O. Melt the gel at 65&#730;C in a heating block.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
-	equal volume of phenol. Mix well with vortex.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
-	at 13,000 rpm for 5 min.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Transfer
-	the supernatant into a new tube and mix with equal volume of
-	phenol/chloroform.</SPAN></FONT></P>
-	<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Perform
-	the same method of DNA precipitation using 2-propanol.</SPAN></FONT></P>
+<br>
+<p><b><font size="3">Blue gel </font></b></p>
+<Table Border Cellspacing="0">
+<Tr><Td>　1xTAE 　</Td><Td>　100mL　</Td></Tr>
+<Tr><Td>　Agarose(Nacalai tesque)　</Td><Td>　1.0g　</Td></Tr>
+<Tr><Td>　Gel indicator(Biodynamics laboratory)　</Td><Td>　200 &micro;L　</Td></Tr>
+</Table>
-Blue gel<br>
+<br>
-・50×TAE 100ml<br>
+<div id="Ligation" style="margin-top:-70px; padding-top:70px;">
-・Agarose(nacalai tesque) 1.0g<br>
+<p><b><font size="5">Ligation</font></b></p>
-・Gel indicator(Biodynamics laboratory) 200ul<br>
+<p>・Dissolve the purified and dried insert DNA in 5µL of H<SUB>2</SUB>O.</p>
-</OL>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
+<p>・Dissolve the purified and dried vector DNA in 5µL of H<SUB>2</SUB>O.</p>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Ligation&gt;</B></SPAN></FONT></P>
+<p>・Mix the two solutions.</p>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
-the purified and dried insert DNA in 5&#181;L of H<SUB>2</SUB>O.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
-the purified and dried vector DNA in 5&#181;L of H2O.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Mix
-the two solutions.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
-&micro;L of Ligation Mix (</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Wako</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)
-to it.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
-for 15 minutes at room temperature.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Transformation&gt;</B></SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
-the ligation solution to competent cells (in a clean bench).</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
-tubes on ice for 20 minutes.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Heat
-shock treatment at 42&#176;C for 35 seconds.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
-tubes on ice for 2 minutes.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
-mL of LB medium into the tube (in a clean bench).</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
-the samples for 20 minutes at 37&#176;C.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Centrifuge
-at 7,000rpm for 3 minutes.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Discard
-the most of supernatant, and spread the remaining cells and LD medium
-in the tube on the plates (in a clean bench).</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
-plates overnight at 37&#176;C.</SPAN></FONT></P>
-<P STYLE="margin-bottom: 0cm"><BR>
-</P>
-</div>
+<p>・Add 10µL of Ligation Mix (Wako) to it.</p>
-</div>
+<p>・Incubate for 15 minutes at room temperature.</p>
+<br>
+<div id="Transformation" style="margin-top:-50px; padding-top:50px;">
+<p><b><font size="5">Transformation</font></b></p>
+<p>・Add the ligation solution to competent cells (in a clean bench).</p>
+<p>・Place tubes on ice for 20 minutes.</p>
+<p>・Heat shock treatment at 42°C for 35 seconds.</p>
+<p>・Place tubes on ice for 2 minutes.</p>
+<p>・Add 1mL of LB medium into the tube (in a clean bench).</p>
+<p>・Incubate the samples for 20 minutes at 37°C.</p>
+<p>・Centrifuge at 7,000rpm for 3 minutes.</p>
+<p>・Discard the most of supernatant, and spread the remaining cells and LD medium in the tube on the plates (in a clean bench).</p>
+<p>・Incubate plates overnight at 37°C.</p>
+</body>
+</html>