Team:METU/Protocols

From 2012.igem.org

(Difference between revisions)
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<html lang="en-US" xmlns="http://www.w3.org/1999/xhtml" dir="ltr"><head>
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<head>
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<meta http-equiv="Content-type" content="text/html; charset=utf-8">
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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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">dNTP</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">dNTP</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
-
<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1 ֌<o:p></o:p></span></p>
+
<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Buffer<span style="mso-spacerun:yes">&nbsp;&nbsp;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Buffer<span style="mso-spacerun:yes">&nbsp;&nbsp;
-
</span><span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>5 ֌<o:p></o:p></span></p>
+
</span><span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">MgCl<sub>2</sub><span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>3
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">MgCl2<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>3
-
֌<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">**Forward Primer <span style="mso-tab-count:
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">**Forward Primer <span style="mso-tab-count:
-
1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0,5 ֌<o:p></o:p></span></p>
+
1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">**Reverse Primer <span style="mso-tab-count:
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">**Reverse Primer <span style="mso-tab-count:
-
1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0,5 ֌<o:p></o:p></span></p>
+
1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Template<span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>-<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Template<span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Taq</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Taq</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
-
Polymerase<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0,2 ֌<o:p></o:p></span></p>
+
Polymerase<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">dH<sub>2</sub>O<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>39,8
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">dH2O<span style="mso-tab-count:2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>39,8
-
֌<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">TOTAL<span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>50
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">TOTAL<span style="mso-tab-count:1">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>50
-
֌<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Use 1ul of 60ng/<span class="SpellE">ul</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">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:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">2.5ul 10x PCR Buffer w/ Mg (1.5mM)<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">0.5ul 25mM MgCl2<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">0.5ul <span class="SpellE">dNTP</span><o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">0.125ul <span class="SpellE">Taq</span><o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">18.375ul sterile water to equal a 25ul <span class="SpellE">rxn</span><o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">18.375ul sterile water to equal a 25ul <span class="SpellE">rxn</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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">95</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">95</span><span style="font-size:12.0pt;
Line 437: Line 436:
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1-5minutes
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">95</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">95</span><span style="font-size:12.0pt;
Line 453: Line 452:
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1min<o:p></o:p></span></p>
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">55</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">55</span><span style="font-size:12.0pt;
Line 465: Line 464:
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1min<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1min<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">72</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">72</span><span style="font-size:12.0pt;
Line 478: Line 477:
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1.5 to
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 1.5 to
2min (usually 2min)<o:p></o:p></span></p>
2min (usually 2min)<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">72</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">72</span><span style="font-size:12.0pt;
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mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C for 10min<o:p></o:p></span></p>
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">4</span><span style="font-size:12.0pt;
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">4</span><span style="font-size:12.0pt;
Line 506: Line 505:
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
mso-hansi-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
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">м/span></span><span style="font-size:
+
symbol;mso-symbol-font-family:Symbol">°</span></span><span style="font-size:
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">C hold<o:p></o:p></span></p>
12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">5 ml LB is mixed with appropriate
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">5 ml LB is mixed with appropriate
Line 532: Line 531:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">A pipette tip is used to pick up a
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubate the culture at 37у for 13-15
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">If the culture becomes saturated: you
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">If the culture becomes saturated: you
-
can <span class="SpellE">reinoculate</span> 30֌ into a new 3mL LB tube<o:p></o:p></span></p>
+
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:&quot;Times New Roman&quot;,&quot;serif&quot;"><o:p>&nbsp;</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:&quot;Times New Roman&quot;,&quot;serif&quot;"><o:p>&nbsp;</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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put 500֌ of a mid-log culture into a
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Put 500µL of a mid-log culture into a
-
1.5mL tube with 500֌ 80% glycerol<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Store at -80C <o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put 50 ֌ and 150 ֌ of whether LB
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spread the cells into the plate until
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Spread the cells into the plate until
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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: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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait until the plates dried.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">After 14-16 hours colonies will show up.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">After 14-16 hours colonies will show up.<o:p></o:p></span></p>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put a drop your culture on the plate<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Put a drop your culture on the plate<o:p></o:p></span></p>
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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: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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Using a pipette <span class="GramE">tip,</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Using a pipette <span class="GramE">tip,</span>
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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: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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait until the plates dried.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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: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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">After 14-16 hours colonies will show up.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">After 14-16 hours colonies will show up.<o:p></o:p></span></p>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">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:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Until <span class="SpellE">agorose</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Until <span class="SpellE">agorose</span>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">After melting, it should be cool enough
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">After melting, it should be cool enough
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 5 ml <span class="SpellE">EtBr</span>.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Then, pour it to container and pay
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Then, pour it to container and pay
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Adjust zoom and position using visible
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Adjust zoom and position using visible
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Before turning on UV load your settings
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Before turning on UV load your settings
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Preview tab, all three options checked<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Active image<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Dynamic integration, auto exposure, 10
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Dynamic integration, auto exposure, 10
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">50/50 brightness/contrast<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Maximize brightness with camera knob
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Maximize brightness with camera knob
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Turn on UV light<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Lower brightness from camera knob if
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Lower brightness from camera knob if
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">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:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait 5 <span class="SpellE">min.and</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait 5 <span class="SpellE">min.and</span>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Resuspended</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Resuspended</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
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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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;"><a href="http://partsregistry.org/Help:Protocols/Competent_Cells" title="Help:Protocols/Competent Cells"><span style="color:windowtext">Competent
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Ice <o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">2ml tube (1 per a transformation')<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">42ۃ water bath<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Petri dishes with LB agar and
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Glass beads or spreader<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">37ۃ incubator<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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:
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Start thawing the competent cells on
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 50 ֌ of thawed competent cells into
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;;background:lightgrey;mso-highlight:lightgrey">Add
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;;background:lightgrey;mso-highlight:lightgrey">Add
-
1 - 2 ֌ of the <span class="SpellE">resuspended</span> DNA to the 2ml tube. Pipet
+
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:&quot;Times New Roman&quot;,&quot;serif&quot;"><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:&quot;Times New Roman&quot;,&quot;serif&quot;"><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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Close tube and incubate the cells on ice
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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:
-
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Heat shock</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Heat shock</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
-
the cells by immersion in a pre-heated water bath at 42ۃ for 60 seconds.<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubate the cells on ice for 5 minutes.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 400 <span class="SpellE">֬</span>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubate the cells at 37ۃ for 2 hours
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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
Line 863: Line 862:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Label two petri dishes with LB agar and
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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 ֬ and 200 ֬ of the transformation onto the
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubate the plate at 37ۃ for 12-14
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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
Line 882: Line 881:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">You can pick a single colony, make a
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">You can pick a single colony, make a
Line 893: Line 892:
115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"> DIGESTION:</span></b><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><br>
115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"> DIGESTION:</span></b><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><br>
<br>
<br>
-
NEB Buffer<span style="mso-spacerun:yes">&nbsp; </span>5 ֬<br>
+
NEB Buffer<span style="mso-spacerun:yes">&nbsp; </span>5 µl<br>
-
BSA<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.5 ֬<br>
+
BSA<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.5 µl<br>
-
Enzyme 1<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.5 ֬<br>
+
Enzyme 1<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.5 µl<br>
-
Enzyme 2<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>0.5 ֬<br>
+
Enzyme 2<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </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:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
Wingdings;mso-ascii-font-family:&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;
mso-bidi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
mso-bidi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;mso-symbol-font-family:
-
Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">༯span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
+
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:&quot;Times New Roman&quot;,&quot;serif&quot;">
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:
-
&quot;Times New Roman&quot;,&quot;serif&quot;">To complete to 50 ֬, add <span class="SpellE">ddH<span style="font-family:&quot;Cambria Math&quot;,&quot;serif&quot;;mso-bidi-font-family:&quot;Cambria Math&quot;">2</span>O</span>.<br>
+
&quot;Times New Roman&quot;,&quot;serif&quot;">To complete to 50 µl, add <span class="SpellE">ddH<span style="font-family:&quot;Cambria Math&quot;,&quot;serif&quot;;mso-bidi-font-family:&quot;Cambria Math&quot;">2</span>O</span>.<br>
-
1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at 37у.<br>
+
1- Keep it in <span class="SpellE">waterbath</span> for 2.5 hours at 37 C0.<br>
2- Run the samples on the gel.<span style="mso-spacerun:yes">&nbsp; </span><o:p></o:p></span></p>
2- Run the samples on the gel.<span style="mso-spacerun:yes">&nbsp; </span><o:p></o:p></span></p>
Line 914: Line 913:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">10X T4 ligase buffer: 2.0 <span class="SpellE">֌</span><o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">6:1 molar ratio of insert to vector
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">6:1 molar ratio of insert to vector
Line 925: Line 924:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add (8.5 - vector and insert volume)<span class="SpellE">֬</span> ddH2O<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">T4 Ligase: 1 <span class="SpellE">֌</span><o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubation at room temperature 1 hour
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubation at room temperature 1 hour
-
and 15 min at 65у for enzyme inactivation.<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Alternatively: Incubation at +
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Alternatively: Incubation at +4°C
overnight.<o:p></o:p></span></p>
overnight.<o:p></o:p></span></p>
Line 951: Line 950:
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Calculating
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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 1,020: Line 1,019:
  <li class="MsoNormal" style="mso-list:l6 level1 lfo19;background:white"><span lang="DE" style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;;
  <li class="MsoNormal" style="mso-list:l6 level1 lfo19;background:white"><span lang="DE" style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;;
     mso-fareast-font-family:&quot;Times New Roman&quot;;mso-ansi-language:DE;mso-fareast-language:
     mso-fareast-font-family:&quot;Times New Roman&quot;;mso-ansi-language:DE;mso-fareast-language:
-
     DE">Solve in ddH<sub>2</sub>0<o:p></o:p></span></li>
+
     DE">Solve in ddH20<o:p></o:p></span></li>
</ul>
</ul>
Line 1,109: Line 1,108:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Take 2 ml cell in to the <span class="SpellE">eppendorf</span> (2ml).<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Discard supernatant.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 2 ml <span class="GramE">cell</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 2 ml <span class="GramE">cell</span>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Discard supernatant.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Resuspend</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Resuspend</span></span><span style="font-size:12.0pt;line-height:115%;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">
-
the cells with 250 ֌ p1* buffer and vortex.<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 250 ֌ P2 buffer and make sure the
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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. DONӔ VORTEX!!!<o:p></o:p></span></p>
+
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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait 5 min at room temperature.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 350 ֌ N3 buffer (cold <span class="GramE">+) inverse</span> gently and immediately.<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 350 µL N3 buffer (cold <span class="GramE">+4C°) 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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Incubate on ice for 15 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 15 min
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 15 min
-
at +.<o:p></o:p></span></p>
+
at +4C°.<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Place the supernatant into the tube that
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Place the supernatant into the tube that
Line 1,181: Line 1,180:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Remove the below part of the tube and
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Remove the below part of the tube and
Line 1,192: Line 1,191:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 500 ֌ PB buffer.<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 750 ֌ PE buffer and wait 1 min near
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 10000g for 1 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 13000g for 1 min
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 13000g for 1 min
Line 1,214: Line 1,213:
<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait 5 min near the flame.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">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:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 50 ֌ elution <span class="GramE">buffer</span>.<o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Wait 1 min near the flame.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Spin down the cells at 13000g for 1 min.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Store your plasmids at+for 1 hour
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">3 volumes of Buffer QG is added to 1
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">1 volume of the INITIAL sample volume of
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Place a <span class="SpellE">QIAquick</span>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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 30ֶ0 <span class="SpellE">s.Discard</span> flow-through. Place the <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 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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 0.75 ml Buffer PE to the <span class="SpellE">QIAquick</span> column with the pipette and centrifuge for 30ֶ0
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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>
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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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Centrifuge the column for an additional
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Centrifuge the column for an additional
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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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Label the top of clean 1.7 ml <span class="SpellE">microcentrifuge</span> tube(s) with the name of your sample(s).
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Label the top of clean 1.7 ml <span class="SpellE">microcentrifuge</span> tube(s) with the name of your sample(s).
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">To elute DNA, add 50 ֬ Buffer EB (10 <span class="SpellE">mM</span> <span class="SpellE">Tris؃l</span>, pH 8.5) to the center
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Detergent-free, sterile glassware and <span class="SpellE">plasticware</span><o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Table-top OD600nm spectrophotometer<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">1 ml DH5 alpha cells<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">LB <o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">0,1M CaCI<sub>2</sub><span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">0,1M CaCI2<span style="mso-spacerun:yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</span><o:p></o:p></span></p>
</span><o:p></o:p></span></p>
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Take 1ml from the DH5 alpha cells which
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Take 1ml from the DH5 alpha cells which
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Divide 100 ml sample into 2 falcon and
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Divide 100 ml sample into 2 falcon and
-
centrifuge for 10 min at 5000 rpm at +.<o:p></o:p></span></p>
+
centrifuge for 10 min at 5000 rpm at +40C.<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Discard the supernatant.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 10 ml 0.1 M cold CaCl<sub>2</sub> into
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 10 ml 0.1 M cold CaCl2 into
supernatant and dissolve the pellet. Put it on ice for 10 min.<o:p></o:p></span></p>
supernatant and dissolve the pellet. Put it on ice for 10 min.<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Centrifuge at 5000 rpm for 5 min at
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Centrifuge at 5000 rpm for 5 min at
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 10 ml CaCl2 and dissolve it by
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Add 10 ml CaCl2 and dissolve it by
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<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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put the sample on ice for half an hour.<o:p></o:p></span></p>
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Centrifuge at 5000 rpm for 5 min at +
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put 2 ml CaCl<sub>2</sub> and dissolve the pellet. <o:p></o:p></span></p>
+
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Put 2 ml CaCl2 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">׼span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
Symbol;mso-bidi-font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
</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:&quot;Times New Roman&quot;,&quot;serif&quot;">Put it on ice for 5 min and keep it at
font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Put it on ice for 5 min and keep it at
-
+.<o:p></o:p></span></p>
+
+40C.<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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-
     16у/30 min, heat kill 80у/20 min<o:p></o:p></span></a></span></li>
+
     16C/30 min, heat kill 80C/20 min<o:p></o:p></span></a></span></li>
  <li class="MsoNormal" style="mso-margin-top-alt:auto;mso-list:l13 level1 lfo15;
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Revision as of 14:34, 25 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)

 

  1. Keep the reagents on ice.
  2. Add the Taq last, and keep it in the freezer until you are ready to add it.
  3. Vortex briefly and quick spin.
  4. 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 C0.
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:

 

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 +4C°) inverse gently and immediately.

·         Incubate on ice for 15 min.

·         Spin down the cells at 10000g for 15 min at +4C°.

·         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 +40C.

·         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 +40C.

 

 

 

11. 3A Assembly Kit:

We used the linear plasmids and the procedure that the iGEM provides us;

 

11.1 Digestion:

 

11.2 Ligation:

 

 

References:12.