Team:METU/Protocols
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<!-- Header --> | <!-- Header --> | ||
<div id="header"> | <div id="header"> | ||
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<li><a href="https://2012.igem.org/Team:METU/Team"><span>Team</span></a></li> | <li><a href="https://2012.igem.org/Team:METU/Team"><span>Team</span></a></li> | ||
- | <li><a href=" | + | <li><a href="#"><span>Project</span></a> |
<div class="dd-holder"> | <div class="dd-holder"> | ||
<div class="dd-t"></div> | <div class="dd-t"></div> | ||
<div class="dd"> | <div class="dd"> | ||
<ul> | <ul> | ||
- | <li><a href="https://2012.igem.org/Team:METU/ | + | <li><a href="https://2012.igem.org/Team:METU/ProjectOverview"><span>Overview</span></a></li> |
- | <li><a href="https://2012.igem.org/Team:METU/CODH"><span> | + | <li><a href="https://2012.igem.org/Team:METU/CODH"><span>CO Converter</span></a></li> |
<li><a href="https://2012.igem.org/Team:METU/KillSwitchOverview"><span>Kill-Switch</span></a></li> | <li><a href="https://2012.igem.org/Team:METU/KillSwitchOverview"><span>Kill-Switch</span></a></li> | ||
<li><a href="https://2012.igem.org/Team:METU/CellLimiter"><span>Cell Limiter</span></a></li> | <li><a href="https://2012.igem.org/Team:METU/CellLimiter"><span>Cell Limiter</span></a></li> | ||
- | <li | + | <li><a href="https://2012.igem.org/Team:METU/datapage"><span>Data Page</span></a></li> |
</ul> | </ul> | ||
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<li><a href="https://2012.igem.org/Team:METU/Results"><span>Results</span></a></li> | <li><a href="https://2012.igem.org/Team:METU/Results"><span>Results</span></a></li> | ||
- | <li><a href=" | + | <li><a href="#"><span>Notebook</span></a> |
<div class="dd-holder"> | <div class="dd-holder"> | ||
<div class="dd-t"></div> | <div class="dd-t"></div> | ||
<div class="dd"> | <div class="dd"> | ||
<ul> | <ul> | ||
- | + | ||
<li><a href="/Team:METU/Timeline">Timeline</a></li> | <li><a href="/Team:METU/Timeline">Timeline</a></li> | ||
- | <li><a href="/Team:METU/Brainstorm"> | + | <li><a href="/Team:METU/Brainstorm">Brainstorming</a></li> |
<li><a href="https://2012.igem.org/Team:METU/Protocols">Protocols</a></li> | <li><a href="https://2012.igem.org/Team:METU/Protocols">Protocols</a></li> | ||
- | + | ||
<li class="last"><a href="/Team:METU/Judging">Judging</a></li> | <li class="last"><a href="/Team:METU/Judging">Judging</a></li> | ||
</ul> | </ul> | ||
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<div id="main"> | <div id="main"> | ||
<div class="shell"> | <div class="shell"> | ||
- | + | <div class="col2"> | |
- | <div | + | <table id="toc" class="toc" style=" |
- | + | width: 400px; | |
+ | "> | ||
<tbody><tr> | <tbody><tr> | ||
<td> | <td> | ||
<div id="table"> | <div id="table"> | ||
<h2><b> Protocols </b></h2> | <h2><b> Protocols </b></h2> | ||
- | <span class="toctoggle"> | + | <span class="toctoggle"> </span></div> |
<ul> | <ul> | ||
<li class="toclevel-1 tocsection-1"><a href="#pcr"><span class="tocnumber">1.</span> <span class="toctext">PCR</span></a></li> | <li class="toclevel-1 tocsection-1"><a href="#pcr"><span class="tocnumber">1.</span> <span class="toctext">PCR</span></a></li> | ||
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</tbody></table> | </tbody></table> | ||
</div> | </div> | ||
- | + | <br></br> | |
<div id="word-id"> | <div id="word-id"> | ||
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<p class="MsoNormal"><span class="GramE"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">1.PCR</span></b></span><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height: | <p class="MsoNormal"><span class="GramE"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">1.PCR</span></b></span><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height: | ||
115%;font-family:"Times New Roman","serif"">:<o:p></o:p></span></b></p> | 115%;font-family:"Times New Roman","serif"">:<o:p></o:p></span></b></p> | ||
+ | |||
+ | |||
<p id="colony" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">1.1<span class="GramE">.Colony</span> PCR:<o:p></o:p></span></b></p> | <p id="colony" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">1.1<span class="GramE">.Colony</span> PCR:<o:p></o:p></span></b></p> | ||
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<p class="MsoListParagraphCxSpFirst" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | </span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | ||
line-height:115%;font-family:"Times New Roman","serif"">dNTP</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | line-height:115%;font-family:"Times New Roman","serif"">dNTP</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | ||
- | <span style="mso-tab-count:2"> </span>1 | + | <span style="mso-tab-count:2"> </span>1 µL<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Buffer<span style="mso-spacerun:yes"> | font-family:"Times New Roman","serif"">Buffer<span style="mso-spacerun:yes"> | ||
- | </span><span style="mso-tab-count:1"> </span>5 | + | </span><span style="mso-tab-count:1"> </span>5 µL<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">MgCl<sub>2</sub><span style="mso-tab-count:2"> </span>3 | font-family:"Times New Roman","serif"">MgCl<sub>2</sub><span style="mso-tab-count:2"> </span>3 | ||
- | + | µL<o:p></o:p></span></p> | |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">**Forward Primer <span style="mso-tab-count: | font-family:"Times New Roman","serif"">**Forward Primer <span style="mso-tab-count: | ||
- | 1"> </span>0,5 | + | 1"> </span>0,5 µL<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">**Reverse Primer <span style="mso-tab-count: | font-family:"Times New Roman","serif"">**Reverse Primer <span style="mso-tab-count: | ||
- | 1"> </span>0,5 | + | 1"> </span>0,5 µL<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Template<span style="mso-tab-count:1"> </span>-<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Template<span style="mso-tab-count:1"> </span>-<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | </span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | ||
line-height:115%;font-family:"Times New Roman","serif"">Taq</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | line-height:115%;font-family:"Times New Roman","serif"">Taq</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | ||
- | Polymerase<span style="mso-tab-count:2"> </span>0,2 | + | Polymerase<span style="mso-tab-count:2"> </span>0,2 µL<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">dH<sub>2</sub>O<span style="mso-tab-count:2"> </span>39,8 | font-family:"Times New Roman","serif"">dH<sub>2</sub>O<span style="mso-tab-count:2"> </span>39,8 | ||
- | + | µL<o:p></o:p></span></p> | |
<p class="MsoListParagraphCxSpLast" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l16 level1 lfo8"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">TOTAL<span style="mso-tab-count:1"> </span>50 | font-family:"Times New Roman","serif"">TOTAL<span style="mso-tab-count:1"> </span>50 | ||
- | + | µL<o:p></o:p></span></p> | |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Use 1ul of 60ng/<span class="SpellE">ul</span> | font-family:"Times New Roman","serif"">Use 1ul of 60ng/<span class="SpellE">ul</span> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Use 1ul of each primer at 3.2pmole/<span class="SpellE">ul</span> concentration or 1.25ul of each primer at 100ng/<span class="SpellE">ul</span> concentration<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Use 1ul of each primer at 3.2pmole/<span class="SpellE">ul</span> concentration or 1.25ul of each primer at 100ng/<span class="SpellE">ul</span> concentration<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">2.5ul 10x PCR Buffer w/ Mg (1.5mM)<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">2.5ul 10x PCR Buffer w/ Mg (1.5mM)<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">0.5ul 25mM MgCl2<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">0.5ul 25mM MgCl2<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">0.5ul <span class="SpellE">dNTP</span><o:p></o:p></span></p> | font-family:"Times New Roman","serif"">0.5ul <span class="SpellE">dNTP</span><o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">0.125ul <span class="SpellE">Taq</span><o:p></o:p></span></p> | font-family:"Times New Roman","serif"">0.125ul <span class="SpellE">Taq</span><o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">18.375ul sterile water to equal a 25ul <span class="SpellE">rxn</span><o:p></o:p></span></p> | font-family:"Times New Roman","serif"">18.375ul sterile water to equal a 25ul <span class="SpellE">rxn</span><o:p></o:p></span></p> | ||
Line 430: | Line 436: | ||
tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">95</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">95</span><span style="font-size:12.0pt; | ||
Line 436: | Line 442: | ||
mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1-5minutes | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1-5minutes | ||
(usually 4min)<o:p></o:p></span></p> | (usually 4min)<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">95</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">95</span><span style="font-size:12.0pt; | ||
Line 452: | Line 458: | ||
mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1min<o:p></o:p></span></p> | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1min<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">55</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">55</span><span style="font-size:12.0pt; | ||
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mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1min<span style="mso-spacerun:yes"> | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1min<span style="mso-spacerun:yes"> | ||
</span>Cycle 30 times<o:p></o:p></span></p> | </span>Cycle 30 times<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">72</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">72</span><span style="font-size:12.0pt; | ||
Line 477: | Line 483: | ||
mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1.5 to | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 1.5 to | ||
2min (usually 2min)<o:p></o:p></span></p> | 2min (usually 2min)<o:p></o:p></span></p> | ||
Line 487: | Line 493: | ||
tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">72</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">72</span><span style="font-size:12.0pt; | ||
Line 493: | Line 499: | ||
mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 10min<o:p></o:p></span></p> | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C for 10min<o:p></o:p></span></p> | ||
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tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | tab-stops:list 1.0in"><!--[if !supportLists]--><span style="font-size:12.0pt; | ||
line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | line-height:115%;font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family: | ||
- | Symbol"><span style="mso-list:Ignore"> | + | Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">4</span><span style="font-size:12.0pt; | font-family:"Times New Roman","serif"">4</span><span style="font-size:12.0pt; | ||
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mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | mso-hansi-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman"; | ||
mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | mso-char-type:symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type: | ||
- | symbol;mso-symbol-font-family:Symbol"> | + | symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size: |
12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C hold<o:p></o:p></span></p> | 12.0pt;line-height:115%;font-family:"Times New Roman","serif"">C hold<o:p></o:p></span></p> | ||
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<p id="liqcul" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | <p id="liqcul" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">5 ml LB is mixed with appropriate | font-family:"Times New Roman","serif"">5 ml LB is mixed with appropriate | ||
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<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">A pipette tip is used to pick up a | font-family:"Times New Roman","serif"">A pipette tip is used to pick up a | ||
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<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Incubate the culture at | + | font-family:"Times New Roman","serif"">Incubate the culture at 37°C for 13-15 |
hours. Do not let the cells become old to not release their plasmids<o:p></o:p></span></p> | hours. Do not let the cells become old to not release their plasmids<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l0 level1 lfo24"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">If the culture becomes saturated: you | font-family:"Times New Roman","serif"">If the culture becomes saturated: you | ||
- | can <span class="SpellE">reinoculate</span> | + | can <span class="SpellE">reinoculate</span> 30µL into a new 3mL LB tube<o:p></o:p></span></p> |
<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-left:.25in"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><o:p> </o:p></span></p> | <p class="MsoNormal" style="mso-margin-top-alt:auto;margin-left:.25in"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><o:p> </o:p></span></p> | ||
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<p id="glystock" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | <p id="glystock" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l3 level1 lfo23"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l3 level1 lfo23"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Put | + | font-family:"Times New Roman","serif"">Put 500µL of a mid-log culture into a |
- | 1.5mL tube with | + | 1.5mL tube with 500µL 80% glycerol<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l3 level1 lfo23"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l3 level1 lfo23"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Store at -80C <o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Store at -80C <o:p></o:p></span></p> | ||
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<p class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Put 50 | + | font-family:"Times New Roman","serif"">Put 50 µL and 150 µL of whether LB |
culture or transformed cells. <o:p></o:p></span></p> | culture or transformed cells. <o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spread the cells into the plate until | font-family:"Times New Roman","serif"">Spread the cells into the plate until | ||
Line 592: | Line 598: | ||
<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait until the plates dried.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Wait until the plates dried.<o:p></o:p></span></p> | ||
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<p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l1 level1 lfo22"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">After 14-16 hours colonies will show up.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">After 14-16 hours colonies will show up.<o:p></o:p></span></p> | ||
Line 612: | Line 618: | ||
<p id="streak" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | <p id="streak" class="MsoListParagraphCxSpFirst" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put a drop your culture on the plate<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Put a drop your culture on the plate<o:p></o:p></span></p> | ||
Line 618: | Line 624: | ||
<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Using a pipette <span class="GramE">tip,</span> | font-family:"Times New Roman","serif"">Using a pipette <span class="GramE">tip,</span> | ||
Line 627: | Line 633: | ||
<p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpMiddle" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait until the plates dried.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Wait until the plates dried.<o:p></o:p></span></p> | ||
Line 633: | Line 639: | ||
<p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | <p class="MsoListParagraphCxSpLast" style="mso-margin-top-alt:auto;mso-add-space: | ||
auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | auto; mso-list:l9 level1 lfo21"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">After 14-16 hours colonies will show up.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">After 14-16 hours colonies will show up.<o:p></o:p></span></p> | ||
Line 651: | Line 657: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Add 0<span class="GramE">,5</span> gr <span class="SpellE">agarose</span> to 50 ml TAE buffer.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Add 0<span class="GramE">,5</span> gr <span class="SpellE">agarose</span> to 50 ml TAE buffer.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Until <span class="SpellE">agorose</span> | font-family:"Times New Roman","serif"">Until <span class="SpellE">agorose</span> | ||
Line 662: | Line 668: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">After melting, it should be cool enough | font-family:"Times New Roman","serif"">After melting, it should be cool enough | ||
Line 668: | Line 674: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Add 5 ml <span class="SpellE">EtBr</span>.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Add 5 ml <span class="SpellE">EtBr</span>.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l17 level1 lfo9"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Then, pour it to container and pay | font-family:"Times New Roman","serif"">Then, pour it to container and pay | ||
Line 685: | Line 691: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Adjust zoom and position using visible | font-family:"Times New Roman","serif"">Adjust zoom and position using visible | ||
Line 691: | Line 697: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Before turning on UV load your settings | font-family:"Times New Roman","serif"">Before turning on UV load your settings | ||
Line 697: | Line 703: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Preview tab, all three options checked<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Preview tab, all three options checked<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Active image<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Active image<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Dynamic integration, auto exposure, 10 | font-family:"Times New Roman","serif"">Dynamic integration, auto exposure, 10 | ||
Line 713: | Line 719: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">50/50 brightness/contrast<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">50/50 brightness/contrast<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Maximize brightness with camera knob | font-family:"Times New Roman","serif"">Maximize brightness with camera knob | ||
Line 724: | Line 730: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Turn on UV light<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Turn on UV light<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l23 level1 lfo6"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Lower brightness from camera knob if | font-family:"Times New Roman","serif"">Lower brightness from camera knob if | ||
Line 744: | Line 750: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l19 level1 lfo13"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l19 level1 lfo13"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">10 <span class="SpellE">ul</span> <span class="SpellE">ddHO</span> is added into the kit plate target part.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">10 <span class="SpellE">ul</span> <span class="SpellE">ddHO</span> is added into the kit plate target part.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l19 level1 lfo13"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l19 level1 lfo13"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait 5 <span class="SpellE">min.and</span> | font-family:"Times New Roman","serif"">Wait 5 <span class="SpellE">min.and</span> | ||
Line 763: | Line 769: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | </span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | ||
line-height:115%;font-family:"Times New Roman","serif"">Resuspended</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | line-height:115%;font-family:"Times New Roman","serif"">Resuspended</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | ||
Line 769: | Line 775: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif""><a href="http://partsregistry.org/Help:Protocols/Competent_Cells" title="Help:Protocols/Competent Cells"><span style="color:windowtext">Competent | font-family:"Times New Roman","serif""><a href="http://partsregistry.org/Help:Protocols/Competent_Cells" title="Help:Protocols/Competent Cells"><span style="color:windowtext">Competent | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Ice <o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Ice <o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">2ml tube (1 per a transformation')<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">2ml tube (1 per a transformation')<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif""> | + | font-family:"Times New Roman","serif"">42ºC water bath<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Petri dishes with LB agar and | font-family:"Times New Roman","serif"">Petri dishes with LB agar and | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Glass beads or spreader<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Glass beads or spreader<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l18 level1 lfo5"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif""> | + | font-family:"Times New Roman","serif"">37ºC incubator<o:p></o:p></span></p> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
Line 811: | Line 817: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Start thawing the competent cells on | font-family:"Times New Roman","serif"">Start thawing the competent cells on | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 50 | + | font-family:"Times New Roman","serif"">Add 50 µL of thawed competent cells into |
pre-chilled 2ml tube. <o:p></o:p></span></p> | pre-chilled 2ml tube. <o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif";background:lightgrey;mso-highlight:lightgrey">Add | font-family:"Times New Roman","serif";background:lightgrey;mso-highlight:lightgrey">Add | ||
- | 1 - 2 | + | 1 - 2 µL of the <span class="SpellE">resuspended</span> DNA to the 2ml tube. Pipet |
up and down a few times, gently. Make sure to keep the competent cells on ice.</span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><o:p></o:p></span></p> | up and down a few times, gently. Make sure to keep the competent cells on ice.</span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Close tube and incubate the cells on ice | font-family:"Times New Roman","serif"">Close tube and incubate the cells on ice | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span class="GramE"><span style="font-size:12.0pt; | </span></span></span><!--[endif]--><span class="GramE"><span style="font-size:12.0pt; | ||
line-height:115%;font-family:"Times New Roman","serif"">Heat shock</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | line-height:115%;font-family:"Times New Roman","serif"">Heat shock</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | ||
- | the cells by immersion in a pre-heated water bath at | + | the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Incubate the cells on ice for 5 minutes.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Incubate the cells on ice for 5 minutes.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 400 <span class="SpellE"> | + | font-family:"Times New Roman","serif"">Add 400 <span class="SpellE">µl</span> |
of LB media (make sure that the broth does not contain antibiotics and is not | of LB media (make sure that the broth does not contain antibiotics and is not | ||
contaminated)<o:p></o:p></span></p> | contaminated)<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Incubate the cells at | + | font-family:"Times New Roman","serif"">Incubate the cells at 37ºC for 2 hours |
while the tubes are rotating or shaking. Important: 2 hour recovery time helps | while the tubes are rotating or shaking. Important: 2 hour recovery time helps | ||
in transformation efficiency, especially for plasmid backbones with antibiotic | in transformation efficiency, especially for plasmid backbones with antibiotic | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Label two petri dishes with LB agar and | font-family:"Times New Roman","serif"">Label two petri dishes with LB agar and | ||
the appropriate antibiotic(s) with the part number, plasmid backbone, and | the appropriate antibiotic(s) with the part number, plasmid backbone, and | ||
- | antibiotic resistance. Plate 20 | + | antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the |
dishes, and spread. This helps ensure that you will be able to pick out a | dishes, and spread. This helps ensure that you will be able to pick out a | ||
single colony.<o:p></o:p></span></p> | single colony.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Incubate the plate at | + | font-family:"Times New Roman","serif"">Incubate the plate at 37ºC for 12-14 |
hours, making sure the agar side of the plate is up. If incubated for too long | hours, making sure the agar side of the plate is up. If incubated for too long | ||
the antibiotics start to break down and un-transformed cells will begin to | the antibiotics start to break down and un-transformed cells will begin to | ||
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<p class="MsoListParagraphCxSpLast" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l21 level1 lfo1"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">You can pick a single colony, make a | font-family:"Times New Roman","serif"">You can pick a single colony, make a | ||
Line 892: | Line 898: | ||
115%;font-family:"Times New Roman","serif""> DIGESTION:</span></b><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><br> | 115%;font-family:"Times New Roman","serif""> DIGESTION:</span></b><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><br> | ||
<br> | <br> | ||
- | NEB Buffer<span style="mso-spacerun:yes"> </span>5 | + | NEB Buffer<span style="mso-spacerun:yes"> </span>5 µl<br> |
- | BSA<span style="mso-spacerun:yes"> </span>0.5 | + | BSA<span style="mso-spacerun:yes"> </span>0.5 µl<br> |
- | Enzyme 1<span style="mso-spacerun:yes"> </span>0.5 | + | Enzyme 1<span style="mso-spacerun:yes"> </span>0.5 µl<br> |
- | Enzyme 2<span style="mso-spacerun:yes"> </span>0.5 | + | Enzyme 2<span style="mso-spacerun:yes"> </span>0.5 µl<br> |
Plasmid* </span><span style="font-size:12.0pt;line-height:115%;font-family: | Plasmid* </span><span style="font-size:12.0pt;line-height:115%;font-family: | ||
Wingdings;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; | Wingdings;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; | ||
mso-bidi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: | mso-bidi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: | ||
- | Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings"> | + | Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> |
1000ng/ml<o:p></o:p></span></p> | 1000ng/ml<o:p></o:p></span></p> | ||
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
- | "Times New Roman","serif"">To complete to 50 | + | "Times New Roman","serif"">To complete to 50 µl, add <span class="SpellE">ddH<span style="font-family:"Cambria Math","serif";mso-bidi-font-family:"Cambria Math"">2</span>O</span>.<br> |
- | 1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at | + | 1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at 37°C.<br> |
2- Run the samples on the gel.<span style="mso-spacerun:yes"> </span><o:p></o:p></span></p> | 2- Run the samples on the gel.<span style="mso-spacerun:yes"> </span><o:p></o:p></span></p> | ||
Line 913: | Line 919: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">10X T4 ligase buffer: 2.0 <span class="SpellE"> | + | font-family:"Times New Roman","serif"">10X T4 ligase buffer: 2.0 <span class="SpellE">µL</span><o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">6:1 molar ratio of insert to vector | font-family:"Times New Roman","serif"">6:1 molar ratio of insert to vector | ||
Line 924: | Line 930: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add (8.5 - vector and insert volume)<span class="SpellE"> | + | font-family:"Times New Roman","serif"">Add (8.5 - vector and insert volume)<span class="SpellE">µl</span> ddH2O<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">T4 Ligase: 1 <span class="SpellE"> | + | font-family:"Times New Roman","serif"">T4 Ligase: 1 <span class="SpellE">µL</span><o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Incubation at room temperature 1 hour | font-family:"Times New Roman","serif"">Incubation at room temperature 1 hour | ||
- | and 15 min at | + | and 15 min at 65°C for enzyme inactivation.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpLast" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l2 level1 lfo10"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Alternatively: Incubation at + | + | font-family:"Times New Roman","serif"">Alternatively: Incubation at +4°C |
overnight.<o:p></o:p></span></p> | overnight.<o:p></o:p></span></p> | ||
Line 950: | Line 956: | ||
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">Calculating | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"">Calculating | ||
Insert Amount:<o:p></o:p></span></b></p> | Insert Amount:<o:p></o:p></span></b></p> | ||
- | <img src="https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png"> | + | <img src="https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png"/> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
Line 1,108: | Line 1,114: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Take 2 ml cell in to the <span class="SpellE">eppendorf</span> (2ml).<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Take 2 ml cell in to the <span class="SpellE">eppendorf</span> (2ml).<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Discard supernatant.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Discard supernatant.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Add 2 ml <span class="GramE">cell</span> | font-family:"Times New Roman","serif"">Add 2 ml <span class="GramE">cell</span> | ||
Line 1,130: | Line 1,136: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Discard supernatant.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Discard supernatant.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | </span></span></span><!--[endif]--><span class="SpellE"><span style="font-size:12.0pt; | ||
line-height:115%;font-family:"Times New Roman","serif"">Resuspend</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | line-height:115%;font-family:"Times New Roman","serif"">Resuspend</span></span><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""> | ||
- | the cells with 250 | + | the cells with 250 µL p1* buffer and vortex.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 250 | + | font-family:"Times New Roman","serif"">Add 250 µL P2 buffer and make sure the |
blue color is equally spread. Gently inverse the <span class="SpellE">eppendorf</span> | blue color is equally spread. Gently inverse the <span class="SpellE">eppendorf</span> | ||
- | 4-5 times. | + | 4-5 times. DON’T VORTEX!!!<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait 5 min at room temperature.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Wait 5 min at room temperature.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 350 | + | font-family:"Times New Roman","serif"">Add 350 µL N3 buffer (cold <span class="GramE">+4°C) inverse</span> gently and immediately.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Incubate on ice for 15 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Incubate on ice for 15 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 15 min | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 15 min | ||
- | at + | + | at +4°C.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Place the supernatant into the tube that | font-family:"Times New Roman","serif"">Place the supernatant into the tube that | ||
Line 1,180: | Line 1,186: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Remove the below part of the tube and | font-family:"Times New Roman","serif"">Remove the below part of the tube and | ||
Line 1,191: | Line 1,197: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 500 | + | font-family:"Times New Roman","serif"">Add 500 µL PB buffer.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 750 | + | font-family:"Times New Roman","serif"">Add 750 µL PE buffer and wait 1 min near |
the flame.<o:p></o:p></span></p> | the flame.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 13000g for 1 min | font-family:"Times New Roman","serif"">Spin down the cells at 13000g for 1 min | ||
Line 1,213: | Line 1,219: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait 5 min near the flame.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Wait 5 min near the flame.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put the filter parts onto the 1<span class="GramE">,5</span> ml <span class="SpellE">eppendorfs</span>.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Put the filter parts onto the 1<span class="GramE">,5</span> ml <span class="SpellE">eppendorfs</span>.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 50 | + | font-family:"Times New Roman","serif"">Add 50 µL elution <span class="GramE">buffer</span>.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Wait 1 min near the flame.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Wait 1 min near the flame.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Spin down the cells at 13000g for 1 min.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Spin down the cells at 13000g for 1 min.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l5 level1 lfo2"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Store your plasmids at+ | + | font-family:"Times New Roman","serif"">Store your plasmids at+4°C for 1 hour |
before further experiments.<o:p></o:p></span></p> | before further experiments.<o:p></o:p></span></p> | ||
Line 1,295: | Line 1,301: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-pagination: | <p class="MsoListParagraphCxSpFirst" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">3 volumes of Buffer QG is added to 1 | font-family:"Times New Roman","serif"">3 volumes of Buffer QG is added to 1 | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | <p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">1 volume of the INITIAL sample volume of | font-family:"Times New Roman","serif"">1 volume of the INITIAL sample volume of | ||
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<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l12 level1 lfo7"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l12 level1 lfo7"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Place a <span class="SpellE">QIAquick</span> | font-family:"Times New Roman","serif"">Place a <span class="SpellE">QIAquick</span> | ||
- | spin column in a provided 2 ml collection tube. Apply the sample to the <span class="SpellE">QIAquick</span> column with the pipette and centrifuge for | + | spin column in a provided 2 ml collection tube. Apply the sample to the <span class="SpellE">QIAquick</span> column with the pipette and centrifuge for 30–60 <span class="SpellE">s.Discard</span> flow-through. Place the <span class="SpellE">QIAquick</span> |
column back into the same tube<o:p></o:p></span></p> | column back into the same tube<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | <p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Add 0.75 ml Buffer PE to the <span class="SpellE">QIAquick</span> column with the pipette and centrifuge for | + | font-family:"Times New Roman","serif"">Add 0.75 ml Buffer PE to the <span class="SpellE">QIAquick</span> column with the pipette and centrifuge for 30–60 |
s. Discard flow-through and place the <span class="SpellE">QIAquick</span> column | s. Discard flow-through and place the <span class="SpellE">QIAquick</span> column | ||
back in the same tube.<o:p></o:p></span></p> | back in the same tube.<o:p></o:p></span></p> | ||
Line 1,324: | Line 1,330: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | <p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Centrifuge the column for an additional | font-family:"Times New Roman","serif"">Centrifuge the column for an additional | ||
Line 1,331: | Line 1,337: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | <p class="MsoListParagraphCxSpMiddle" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Label the top of clean 1.7 ml <span class="SpellE">microcentrifuge</span> tube(s) with the name of your sample(s). | font-family:"Times New Roman","serif"">Label the top of clean 1.7 ml <span class="SpellE">microcentrifuge</span> tube(s) with the name of your sample(s). | ||
Line 1,338: | Line 1,344: | ||
<p class="MsoListParagraphCxSpLast" style=" mso-pagination: | <p class="MsoListParagraphCxSpLast" style=" mso-pagination: | ||
none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | none;mso-list:l12 level1 lfo7;mso-layout-grid-align:none;text-autospace:none"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">To elute DNA, add 50 | + | font-family:"Times New Roman","serif"">To elute DNA, add 50 µl Buffer EB (10 <span class="SpellE">mM</span> <span class="SpellE">Tris·Cl</span>, pH 8.5) to the center |
of the <span class="SpellE">QIAquick</span> membrane. Let it stand for 1 min and centrifuge | of the <span class="SpellE">QIAquick</span> membrane. Let it stand for 1 min and centrifuge | ||
the column for 1 min. <o:p></o:p></span></p> | the column for 1 min. <o:p></o:p></span></p> | ||
Line 1,353: | Line 1,359: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Detergent-free, sterile glassware and <span class="SpellE">plasticware</span><o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Detergent-free, sterile glassware and <span class="SpellE">plasticware</span><o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Table-top OD600nm spectrophotometer<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Table-top OD600nm spectrophotometer<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">1 ml DH5 alpha cells<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">1 ml DH5 alpha cells<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">LB <o:p></o:p></span></p> | font-family:"Times New Roman","serif"">LB <o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l15 level1 lfo4"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">0,1M CaCI<sub>2</sub><span style="mso-spacerun:yes"> | font-family:"Times New Roman","serif"">0,1M CaCI<sub>2</sub><span style="mso-spacerun:yes"> | ||
Line 1,381: | Line 1,387: | ||
<p class="MsoListParagraphCxSpFirst" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpFirst" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Take 1ml from the DH5 alpha cells which | font-family:"Times New Roman","serif"">Take 1ml from the DH5 alpha cells which | ||
Line 1,390: | Line 1,396: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Divide 100 ml sample into 2 falcon and | font-family:"Times New Roman","serif"">Divide 100 ml sample into 2 falcon and | ||
- | centrifuge for 10 min at 5000 rpm at + | + | centrifuge for 10 min at 5000 rpm at +4°C.<o:p></o:p></span></p> |
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Discard the supernatant.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Discard the supernatant.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Add 10 ml 0.1 M cold CaCl<sub>2</sub> into | font-family:"Times New Roman","serif"">Add 10 ml 0.1 M cold CaCl<sub>2</sub> into | ||
Line 1,407: | Line 1,413: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Centrifuge at 5000 rpm for 5 min at | font-family:"Times New Roman","serif"">Centrifuge at 5000 rpm for 5 min at | ||
Line 1,413: | Line 1,419: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Add 10 ml CaCl2 and dissolve it by | font-family:"Times New Roman","serif"">Add 10 ml CaCl2 and dissolve it by | ||
Line 1,419: | Line 1,425: | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put the sample on ice for half an hour.<o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Put the sample on ice for half an hour.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">Centrifuge at 5000 rpm for 5 min at + | + | font-family:"Times New Roman","serif"">Centrifuge at 5000 rpm for 5 min at +4°C |
.Then, discard the supernatant.<o:p></o:p></span></p> | .Then, discard the supernatant.<o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpMiddle" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put 2 ml CaCl<sub>2</sub> and dissolve the pellet. <o:p></o:p></span></p> | font-family:"Times New Roman","serif"">Put 2 ml CaCl<sub>2</sub> and dissolve the pellet. <o:p></o:p></span></p> | ||
<p class="MsoListParagraphCxSpLast" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | <p class="MsoListParagraphCxSpLast" style=" mso-list:l4 level1 lfo3"><!--[if !supportLists]--><span style="font-size:12.0pt;line-height:115%;font-family:Symbol;mso-fareast-font-family: | ||
- | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore"> | + | Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman""> |
</span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | </span></span></span><!--[endif]--><span style="font-size:12.0pt;line-height:115%; | ||
font-family:"Times New Roman","serif"">Put it on ice for 5 min and keep it at | font-family:"Times New Roman","serif"">Put it on ice for 5 min and keep it at | ||
- | + | + | +4°C.<o:p></o:p></span></p> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
Line 1,478: | Line 1,484: | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif"">0.5 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span class="SpellE | + | font-family:"Times New Roman","serif"">0.5 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span class="SpellE"><span style="color:windowtext">EcoRI</span></span><span class="MsoHyperlink"><span style="color:windowtext">-HF</span></span></span></li> |
<span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"; | <span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif"; | ||
mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin; | mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin; | ||
Line 1,484: | Line 1,490: | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""> <span style="color:windowtext;text-decoration:none;text-underline:none">0.5 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span class="SpellE"><u>PstI</u></span></span><o:p></o:p></span></a></span></li> |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">0.5 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span class="SpellE"><span style="<u>DpnI</u></span></span><span class="apple-converted-space"> </span>(Used to digest any template DNA |
from production)<o:p></o:p></span></a></span></li> | from production)<o:p></o:p></span></a></span></li> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">18 <span class="SpellE">ul</span> dH20<o:p></o:p></span></a></span></li> |
</ul> | </ul> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level1 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level1 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Digest |
Plasmid Backbone<o:p></o:p></span></a></span></li> | Plasmid Backbone<o:p></o:p></span></a></span></li> | ||
<ul type="circle"> | <ul type="circle"> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add 4 <span class="SpellE">ul</span> linearized plasmid backbone (25ng/<span class="SpellE">ul</span> for 100ng total)<o:p></o:p></span></a></span></li> |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add 4 <span class="SpellE">ul</span> of Enzyme Master Mix<o:p></o:p></span></a></span></li> |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Digest |
37C/30 min, heat kill 80C/20 min<o:p></o:p></span></a></span></li> | 37C/30 min, heat kill 80C/20 min<o:p></o:p></span></a></span></li> | ||
</ul> | </ul> | ||
Line 1,512: | Line 1,518: | ||
<p class="MsoNormal" style="mso-margin-top-alt:auto;background:white"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height: | <p class="MsoNormal" style="mso-margin-top-alt:auto;background:white"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height: | ||
- | 115%;font-family:"Times New Roman","serif" | + | 115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></b></p> |
- | <p id="lig" class="MsoNormal"><span class="mw-headline"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif" | + | <p id="lig" class="MsoNormal"><span class="mw-headline"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">11.2 Ligation:</span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif"; |
mso-ascii-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family: | mso-ascii-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family: | ||
"Times New Roman";mso-bidi-theme-font:minor-bidi;color:windowtext;text-decoration: | "Times New Roman";mso-bidi-theme-font:minor-bidi;color:windowtext;text-decoration: | ||
Line 1,522: | Line 1,528: | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add 2ul |
of digested plasmid backbone (25 <span class="SpellE">ng</span>)<o:p></o:p></span></a></span></li> | of digested plasmid backbone (25 <span class="SpellE">ng</span>)<o:p></o:p></span></a></span></li> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add <span class="SpellE">equimolar</span> amount of <span class="SpellE">EcoRI</span>-HF |
<span class="SpellE">SpeI</span> digested fragment (< 3 <span class="SpellE">ul</span>)<o:p></o:p></span></a></span></li> | <span class="SpellE">SpeI</span> digested fragment (< 3 <span class="SpellE">ul</span>)<o:p></o:p></span></a></span></li> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add <span class="SpellE">equimolar</span> amount of <span class="SpellE">XbaI</span> <span class="SpellE">PstI</span> digested fragment (< 3 <span class="SpellE">ul</span>)<o:p></o:p></span></a></span></li> |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add 1 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span style="mso-field-code:""><u>T4 |
DNA ligase buffer</u></span>.<o:p></o:p></span></a></span></li> | DNA ligase buffer</u></span>.<o:p></o:p></span></a></span></li> | ||
<ul type="circle"> | <ul type="circle"> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level2 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level2 lfo15; | ||
- | background:white"><span class="apple-converted-space"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif" | + | background:white"><span class="apple-converted-space"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"> </span><b><span style="color:windowtext;text-decoration:none;text-underline:none">Note:</span></b><span style="color:windowtext;text-decoration:none;text-underline:none"> </span><span style="color:windowtext;text-decoration:none;text-underline:none">Do not |
use quick ligase</span><span style="color:windowtext;text-decoration: | use quick ligase</span><span style="color:windowtext;text-decoration: | ||
none;text-underline:none"><o:p></o:p></span></a></span></span></li> | none;text-underline:none"><o:p></o:p></span></a></span></span></li> | ||
Line 1,543: | Line 1,549: | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif"">Add 0.5 <span class="SpellE">ul</span><span class="apple-converted-space"> </span><span style="mso-field-code:" "><u>T4 |
DNA ligase</u></span><o:p></o:p></span></a></span></li> | DNA ligase</u></span><o:p></o:p></span></a></span></li> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Add |
water to 10 <span class="SpellE">ul</span><o:p></o:p></span></a></span></li> | water to 10 <span class="SpellE">ul</span><o:p></o:p></span></a></span></li> | ||
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Ligate |
- | + | 16°C/30 min, heat kill 80°C/20 min<o:p></o:p></span></a></span></li> | |
<li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15; | ||
background:white"><span style="font-size:12.0pt;line-height:115%; | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
- | font-family:"Times New Roman","serif" | + | font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">Transform |
with 1-2 <span class="SpellE">ul</span> of product<o:p></o:p></span></a></span></li> | with 1-2 <span class="SpellE">ul</span> of product<o:p></o:p></span></a></span></li> | ||
</ul> | </ul> | ||
<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-left:.25in;background: | <p class="MsoNormal" style="mso-margin-top-alt:auto;margin-left:.25in;background: | ||
- | white"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif" | + | white"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
- | "Times New Roman","serif" | + | "Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> |
- | <p id="ref" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif" | + | <p id="ref" class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none">References:<o:p></o:p></span></a>12.</span></b></p> |
- | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif" | + | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></b></p> |
<p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | <p class="MsoNormal"><span style="font-size:12.0pt;line-height:115%;font-family: | ||
- | "Times New Roman","serif""><a href="http://www. | + | "Times New Roman","serif""><span style="color:windowtext;text-decoration:none;text-underline:none"><o:p> </o:p></span></a></span></p> |
+ | |||
+ | |||
+ | <p><ul type="circle"> | ||
+ | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
+ | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
+ | font-family:"Times New Roman","serif""><a href="http://www.qiagen.com" title="#"><span style="color:windowtext;text-decoration:none;text-underline:none">http://www.qiagen.com</span></a></span></li> | ||
+ | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
+ | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
+ | font-family:"Times New Roman","serif""><a href="http://www.neb.com/nebecomm/products/productR0176.asp" title="#"><span style="color:windowtext;text-decoration:none;text-underline:none">http://www.neb.com/nebecomm/products/productR0176.asp<o:p></o:p></span></a></span></li> | ||
+ | <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l10 level2 lfo16; | ||
+ | background:white"><span style="font-size:12.0pt;line-height:115%; | ||
+ | font-family:"Times New Roman","serif""><a href="http://partsregistry.org/Help:3A_Assembly_Kit" title="#"><span style="color:windowtext;text-decoration:none;text-underline:none">http://partsregistry.org/Help:3A_Assembly_Kit</span></a></span></li> | ||
+ | </ul> | ||
+ | </p> | ||
</div> | </div> | ||
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Latest revision as of 22:48, 26 September 2012
Protocols |
1.PCR:
1.1.Colony PCR:
Prepare mastermix
according to your sample amount (except Taq and
template)
(For 1x sample preparation)
·
dNTP
1 µL
·
Buffer
5 µL
·
MgCl2 3
µL
·
**Forward Primer 0,5 µL
·
**Reverse Primer 0,5 µL
·
Template -
·
Taq
Polymerase 0,2 µL
·
dH2O 39,8
µL
·
TOTAL 50
µL
* You should add ingredients from largest amount to
smallest amount.
* Before addition of primers and template you can do
vortex.
** First you should pour ddH20 onto dried primers
according to the amount written in the primer sheet then you should dilute
(1:10) it in to new eppendorf. (10 ml primer+ 90 ml
ddH20)
Pour your samples into PCR tubes and add template
that you pick from the petri with toothpick or tip and finally add taq polymerase.
Then place your samples into the PCR machine and do
regular PCR.
1.2
PCR:
For a 25ul rxn:
·
Use 1ul of 60ng/ul
or 100ng/ul DNA
·
Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each primer at 100ng/ul concentration
·
2.5ul 10x PCR Buffer w/ Mg (1.5mM)
·
0.5ul 25mM MgCl2
·
0.5ul dNTP
·
0.125ul Taq
·
18.375ul sterile water to equal a 25ul rxn
(*if not making master mix, dilute Taq so that
you can add 1ul of Taq and 17.5ul
sterile water to equal a 25ul rxn)
For a 50ul rxn:
- Use
2ul of 60ng/ul or 100ng/ul
DNA
- Use
2ul of each primer at 3.2pmole/ul concentration
or 2.5ul of each primer at 100ng/ul
concentration
- 5ul
10x PCR Buffer w/ Mg
- 1ul
25mM MgCl2
- 1ul
dNTP
- 0.25ul
Taq
- 36.75ul
sterile water to equal a 50ul rxn
(*if not making a master mix, dilute Taq so
that you can add 1ul of Taq and 36ul
sterile water to equal a 50ul rxn)
- Keep
the reagents on ice.
- Add
the Taq last, and keep it in the freezer until
you are ready to add it.
- Vortex
briefly and quick spin.
- Cycle:
·
95°C for 1-5minutes
(usually 4min)
·
95°C for 1min
·
55°C for 1min
Cycle 30 times
·
72°C for 1.5 to
2min (usually 2min)
·
72°C for 10min
·
4°C hold
2.CULTURES:
2.1 Liquid Culture:
·
5 ml LB is mixed with appropriate
antibiotic and a culture tube is prepared with certain labels.
·
A pipette tip is used to pick up a
colony from the plate and release it into the LB by stirring and pipetting up
and down
·
Incubate the culture at 37°C for 13-15
hours. Do not let the cells become old to not release their plasmids
·
If the culture becomes saturated: you
can reinoculate 30µL into a new 3mL LB tube
2.2 Glycerol Stock:
·
Put 500µL of a mid-log culture into a
1.5mL tube with 500µL 80% glycerol
·
Store at -80C
2.3 Spreading Plates:
·
Put 50 µL and 150 µL of whether LB
culture or transformed cells.
·
Spread the cells into the plate until
there is no seen liquid.
·
Wait until the plates dried.
·
After 14-16 hours colonies will show up.
2.4 Streaking Plates:
·
Put a drop your culture on the plate
·
Using a pipette tip,
spread the drop out into a zigzag. Then use one edge of the zigzag to draw out
another zigzag. Repeat to have about 3 zigzags (this makes the culture get
spread out more and more with each streak)
·
Wait until the plates dried.
·
After 14-16 hours colonies will show up.
3.GELS:
3.1
Gel Preparation (%1 gel)
·
Add 0,5 gr agarose to 50 ml TAE buffer.
·
Until agorose
being melted, heat it in microwave and do not forget to stir it often.
·
After melting, it should be cool enough
to handle it.
·
Add 5 ml EtBr.
·
Then, pour it to container and pay
attention not to form bubbles in gel.
3.2
Gel Photo Imaging:
·
Adjust zoom and position using visible
light
·
Before turning on UV load your settings
file which has the following parameters:
·
Preview tab, all three options checked
·
Active image
·
Dynamic integration, auto exposure, 10
frames
·
50/50 brightness/contrast
·
Maximize brightness with camera knob
(counterclockwise)
·
Turn on UV light
·
Lower brightness from camera knob if
necessary
4.GETTING THE DNA PARTS FROM KIT PLATE:
·
10 ul ddHO is added into the kit plate target part.
·
Wait 5 min.and
get the part by well pipetting.
5.TRANSFORMATION:
Materials:
·
Resuspended
DNA (2 ul )
·
Competent
cells (50ul per transformation)
·
Ice
·
2ml tube (1 per a transformation')
·
42ºC water bath
·
Petri dishes with LB agar and
appropriate antibiotic (2 or 3 per transformation)
·
Glass beads or spreader
·
37ºC incubator
Preparation:
·
Start thawing the competent cells on
ice.
·
Add 50 µL of thawed competent cells into
pre-chilled 2ml tube.
·
Add
1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet
up and down a few times, gently. Make sure to keep the competent cells on ice.
·
Close tube and incubate the cells on ice
for 30 minutes.
·
Heat shock
the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
·
Incubate the cells on ice for 5 minutes.
·
Add 400 µl
of LB media (make sure that the broth does not contain antibiotics and is not
contaminated)
·
Incubate the cells at 37ºC for 2 hours
while the tubes are rotating or shaking. Important: 2 hour recovery time helps
in transformation efficiency, especially for plasmid backbones with antibiotic
resistance other than ampicillin.
·
Label two petri dishes with LB agar and
the appropriate antibiotic(s) with the part number, plasmid backbone, and
antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the
dishes, and spread. This helps ensure that you will be able to pick out a
single colony.
·
Incubate the plate at 37ºC for 12-14
hours, making sure the agar side of the plate is up. If incubated for too long
the antibiotics start to break down and un-transformed cells will begin to
grow. This is especially true for ampicillin - because the resistance enzyme is
excreted by the bacteria, and inactivates the antibiotic outside of the
bacteria.
·
You can pick a single colony, make a
glycerol stock, grow up a cell culture.
6.RESTRICTION DIGESTION:
NEB Buffer 5 µl
BSA 0.5 µl
Enzyme 1 0.5 µl
Enzyme 2 0.5 µl
Plasmid* à
1000ng/ml
To complete to 50 µl, add ddH2O.
1- Keep it in waterbath for 2.5 hours at 37°C.
2- Run the samples on the gel.
7.LIGATION:
·
10X T4 ligase buffer: 2.0 µL
·
6:1 molar ratio of insert to vector
(~10ng vector)
·
Add (8.5 - vector and insert volume)µl ddH2O
·
T4 Ligase: 1 µL
·
Incubation at room temperature 1 hour
and 15 min at 65°C for enzyme inactivation.
·
Alternatively: Incubation at +4°C
overnight.
Calculating
Insert Amount:
![](https://static.igem.org/mediawiki/2012/c/cb/K%C3%BCbra.png)
8.MATERIALS AND CHEMICALS:
8.1 TAE Buffer:
For 1 L of 50 x TAE buffer you need:
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- Adjust pH to 7.8
- Solve in dH2O
20 mL of the stock is diluted in 1 L
dH2O for the gel electrophoresis.
8.2 DNA Loading Buffer
- 50 % (v/v) glycerol
- 1 mM EDTA
- 0.1 % (w/v) bromphenol blue
- Solve in ddH20
8.3 LB Medium
For 1 L of LB medium you need:
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g
Agar-Agar (for plates)
- Adjust pH to 7.0
8.4
LB Agar
For 1 L of LB Agar you need
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g
Agar-Agar (for plates)
- Adjust pH to 7.0
9.QIAGEN Kits
9.1
Plasmid Isolation:
Usually there is a protocol in the kit. First, you should
read it.
·
Take 2 ml cell in to the eppendorf (2ml).
·
Spin down the cells at 10000g for 1 min.
·
Discard supernatant.
·
Add 2 ml cell
in to the eppendorf again, totally 4 ml will be
pelleted.
·
Spin down the cells at 10000g for 1 min.
·
Discard supernatant.
·
Resuspend
the cells with 250 µL p1* buffer and vortex.
·
Add 250 µL P2 buffer and make sure the
blue color is equally spread. Gently inverse the eppendorf
4-5 times. DON’T VORTEX!!!
·
Wait 5 min at room temperature.
·
Add 350 µL N3 buffer (cold +4°C) inverse gently and immediately.
·
Incubate on ice for 15 min.
·
Spin down the cells at 10000g for 15 min
at +4°C.
·
Place the supernatant into the tube that
is placed into the kit.
·
Spin down the cells at 10000g for 1 min.
·
Remove the below part of the tube and
discard supernatant.
·
Add 500 µL PB buffer.
·
Add 750 µL PE buffer and wait 1 min near
the flame.
·
Spin down the cells at 10000g for 1 min.
·
Spin down the cells at 13000g for 1 min
again.
·
Wait 5 min near the flame.
·
Put the filter parts onto the 1,5 ml eppendorfs.
·
Add 50 µL elution buffer.
·
Wait 1 min near the flame.
·
Spin down the cells at 13000g for 1 min.
·
Store your plasmids at+4°C for 1 hour
before further experiments.
9.2
Gel Extraction:
We used QIAGEN kit do with the following
changes:
http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf
Changes
in that protocol:
At step 0: Wear
disposable lab coat or long sleeves to prevent UV-burn. Use a razor
blade/scalpel to excise the band.
At step 4: Add 10 ul for
3M NaOAc no matter if the color is yellow or not.
This will correct the pH and should turn the QG back to yellow.
At step 7: Combine samples of
identical DNA in the same column (spin multiple times if necessary).
At step 13: Elute in 30 ul
rather than 50 ul to really maximize yield.
9.3
PCR Purification
We used the protocol provided with the QIAGEN kit.
·
3 volumes of Buffer QG is added to 1
volume of the PCR sample and mixed by vortexing.
·
1 volume of the INITIAL sample volume of
isopropanol is added to the sample and mix.
·
Place a QIAquick
spin column in a provided 2 ml collection tube. Apply the sample to the QIAquick column with the pipette and centrifuge for 30–60 s.Discard flow-through. Place the QIAquick
column back into the same tube
·
Add 0.75 ml Buffer PE to the QIAquick column with the pipette and centrifuge for 30–60
s. Discard flow-through and place the QIAquick column
back in the same tube.
·
Centrifuge the column for an additional
1 min.
·
Label the top of clean 1.7 ml microcentrifuge tube(s) with the name of your sample(s).
Transfer QIAquick column(s) to the tube(s).
·
To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center
of the QIAquick membrane. Let it stand for 1 min and centrifuge
the column for 1 min.
10.Competent Cells:
Materials:
·
Detergent-free, sterile glassware and plasticware
·
Table-top OD600nm spectrophotometer
·
1 ml DH5 alpha cells
·
LB
·
0,1M CaCI2
Preparation:
·
Take 1ml from the DH5 alpha cells which
were grown one day ago and put it in to 100ml LB medium. Keep it at 370C for 2
hours and measure it by spectrophotometer at 600 nm wavelength until absorbance
reaches OD of
0.375.
·
Divide 100 ml sample into 2 falcon and
centrifuge for 10 min at 5000 rpm at +4°C.
·
Discard the supernatant.
·
Add 10 ml 0.1 M cold CaCl2 into
supernatant and dissolve the pellet. Put it on ice for 10 min.
·
Centrifuge at 5000 rpm for 5 min at
+40C. Then, discard the supernatant.
·
Add 10 ml CaCl2 and dissolve it by
shaking it up and down.
·
Put the sample on ice for half an hour.
·
Centrifuge at 5000 rpm for 5 min at +4°C
.Then, discard the supernatant.
·
Put 2 ml CaCl2 and dissolve the pellet.
·
Put it on ice for 5 min and keep it at
+4°C.
11.
3A Assembly Kit:
We used the linear plasmids and the procedure that
the iGEM provides us;
11.1
Digestion:
- Enzyme Master Mix for Plasmid
Backbone (25ul total, for 6 rxns)
- 5 ul NEB
Buffer 2
- 0.5 ul
BSA
- 0.5 ul EcoRI-HF
- 0.5 ul PstI
- 0.5 ul (Used to digest any template DNA
from production)
- 18 ul dH20
- Digest
Plasmid Backbone
- Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
- Add 4 ul of Enzyme Master Mix
- Digest
37C/30 min, heat kill 80C/20 min
11.2 Ligation:
- Add 2ul
of digested plasmid backbone (25 ng)
- Add equimolar amount of EcoRI-HF
SpeI digested fragment (< 3 ul)
- Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
- Add 1 ul T4
DNA ligase buffer.
- Note: Do not
use quick ligase
- Add 0.5 ul T4
DNA ligase
- Add
water to 10 ul
- Ligate
16°C/30 min, heat kill 80°C/20 min
- Transform
with 1-2 ul of product
References: