User:Neil J. Parikh/Lab Notebook: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
<html> | |||
<html | |||
<head> | <head> | ||
<meta http-equiv=Content-Type content="text/html; charset=windows-1252"> | <meta http-equiv=Content-Type content="text/html; charset=windows-1252"> | ||
<meta name=Generator content="Microsoft Word 12 (filtered)"> | |||
<meta name=Generator content="Microsoft Word 12 | |||
<title>This is an experiment title</title> | <title>This is an experiment title</title> | ||
<style> | <style> | ||
<!-- | <!-- | ||
| Line 348: | Line 10: | ||
@font-face | @font-face | ||
{font-family:Wingdings; | {font-family:Wingdings; | ||
panose-1:5 0 0 0 0 0 0 0 0 | panose-1:5 0 0 0 0 0 0 0 0 0;} | ||
@font-face | @font-face | ||
{font-family:"Cambria Math"; | {font-family:"Cambria Math"; | ||
panose-1:2 4 5 3 5 4 6 3 2 4 | panose-1:2 4 5 3 5 4 6 3 2 4;} | ||
@font-face | @font-face | ||
{font-family:Cambria; | {font-family:Cambria; | ||
panose-1:2 4 5 3 5 4 6 3 2 4 | panose-1:2 4 5 3 5 4 6 3 2 4;} | ||
@font-face | @font-face | ||
{font-family:Calibri; | {font-family:Calibri; | ||
panose-1:2 15 5 2 2 2 4 3 2 4 | panose-1:2 15 5 2 2 2 4 3 2 4;} | ||
@font-face | @font-face | ||
{font-family: | {font-family:Times; | ||
panose-1:2 2 6 3 5 4 5 2 3 4;} | |||
@font-face | @font-face | ||
{font-family: | {font-family:"Lucida Grande";} | ||
/* Style Definitions */ | /* Style Definitions */ | ||
p.MsoNormal, li.MsoNormal, div.MsoNormal | p.MsoNormal, li.MsoNormal, div.MsoNormal | ||
{ | {margin:0in; | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Arial","sans-serif | font-family:"Arial","sans-serif";} | ||
h1 | h1 | ||
{ | {margin-top:.25in; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:6.0pt; | margin-bottom:6.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
page-break-after:avoid; | page-break-after:avoid; | ||
background:silver; | background:silver; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif" | |||
h2 | h2 | ||
{ | {margin-top:24.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:3.0pt; | margin-bottom:3.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
page-break-after:avoid; | page-break-after:avoid; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif" | |||
p.MsoHeader, li.MsoHeader, div.MsoHeader | p.MsoHeader, li.MsoHeader, div.MsoHeader | ||
{ | {margin-top:0in; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:6.0pt; | margin-bottom:6.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
border:none; | border:none; | ||
padding:0in; | padding:0in; | ||
font-size:9.0pt; | font-size:9.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold; | font-weight:bold; | ||
font-style:italic;} | |||
font-style:italic | |||
p.MsoFooter, li.MsoFooter, div.MsoFooter | p.MsoFooter, li.MsoFooter, div.MsoFooter | ||
{ | {margin-top:0in; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:6.0pt; | margin-bottom:6.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
border:none; | border:none; | ||
padding:0in; | padding:0in; | ||
font-size:9.0pt; | font-size:9.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold; | font-weight:bold; | ||
font-style:italic;} | |||
font-style:italic | |||
p.MsoListBullet, li.MsoListBullet, div.MsoListBullet | p.MsoListBullet, li.MsoListBullet, div.MsoListBullet | ||
{ | {margin-top:6.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:0in; | margin-bottom:0in; | ||
| Line 494: | Line 77: | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
text-indent:-.25in; | text-indent:-.25in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
p.MsoListNumber, li.MsoListNumber, div.MsoListNumber | p.MsoListNumber, li.MsoListNumber, div.MsoListNumber | ||
{ | {margin-top:6.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:0in; | margin-bottom:0in; | ||
| Line 511: | Line 86: | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
text-indent:-.25in; | text-indent:-.25in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
p.MsoListBullet2, li.MsoListBullet2, div.MsoListBullet2 | p.MsoListBullet2, li.MsoListBullet2, div.MsoListBullet2 | ||
{ | {margin-top:0in; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:0in; | margin-bottom:0in; | ||
| Line 529: | Line 95: | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
text-indent:-.25in; | text-indent:-.25in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
p.MsoTitle, li.MsoTitle, div.MsoTitle | p.MsoTitle, li.MsoTitle, div.MsoTitle | ||
{ | {margin-top:3.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:3.0pt; | margin-bottom:3.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold;} | |||
font-weight:bold | |||
p.MsoBodyText, li.MsoBodyText, div.MsoBodyText | p.MsoBodyText, li.MsoBodyText, div.MsoBodyText | ||
{ | {margin-top:0in; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:6.0pt; | margin-bottom:6.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
p.MsoSubtitle, li.MsoSubtitle, div.MsoSubtitle | p.MsoSubtitle, li.MsoSubtitle, div.MsoSubtitle | ||
{ | {mso-style-link:"Subtitle Char"; | ||
margin-top:0in; | margin-top:0in; | ||
margin-right:0in; | margin-right:0in; | ||
| Line 580: | Line 119: | ||
margin-left:0in; | margin-left:0in; | ||
text-align:center; | text-align:center; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Cambria","serif | font-family:"Cambria","serif";} | ||
a:link, span.MsoHyperlink | a:link, span.MsoHyperlink | ||
{ | {color:blue; | ||
text-decoration:underline;} | |||
text-decoration:underline | |||
a:visited, span.MsoHyperlinkFollowed | a:visited, span.MsoHyperlinkFollowed | ||
{ | {color:purple; | ||
text-decoration:underline;} | |||
text-decoration:underline | |||
p.MsoAcetate, li.MsoAcetate, div.MsoAcetate | p.MsoAcetate, li.MsoAcetate, div.MsoAcetate | ||
{ | {margin:0in; | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
font-size:9.0pt; | font-size:9.0pt; | ||
font-family:"Lucida Grande"; | font-family:"Lucida Grande";} | ||
p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph | |||
{margin-top:0in; | |||
margin-right:0in; | |||
margin-bottom:0in; | |||
margin-left:.5in; | |||
margin-bottom:.0001pt; | |||
font-size:12.0pt; | |||
font-family:"Arial","sans-serif";} | |||
span.MsoIntenseEmphasis | span.MsoIntenseEmphasis | ||
{ | {color:#4F81BD; | ||
font-weight:bold; | font-weight:bold; | ||
font-style:italic;} | font-style:italic;} | ||
p.ExperimentNumber, li.ExperimentNumber, div.ExperimentNumber | p.ExperimentNumber, li.ExperimentNumber, div.ExperimentNumber | ||
{mso-style-name:"Experiment Number"; | {mso-style-name:"Experiment Number"; | ||
margin-top:3.0pt; | margin-top:3.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
| Line 626: | Line 151: | ||
margin-left:0in; | margin-left:0in; | ||
text-align:right; | text-align:right; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold; | font-weight:bold; | ||
font-style:italic;} | |||
font-style:italic | |||
p.BodyTextFirst, li.BodyTextFirst, div.BodyTextFirst | p.BodyTextFirst, li.BodyTextFirst, div.BodyTextFirst | ||
{mso-style-name:"Body Text First"; | {mso-style-name:"Body Text First"; | ||
margin-top:6.0pt; | margin-top:6.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:6.0pt; | margin-bottom:6.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
p.MaterialsListFirst, li.MaterialsListFirst, div.MaterialsListFirst | p.MaterialsListFirst, li.MaterialsListFirst, div.MaterialsListFirst | ||
{mso-style-name:"Materials List First"; | {mso-style-name:"Materials List First"; | ||
margin-top:6.0pt; | margin-top:6.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
| Line 663: | Line 171: | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
text-indent:-.25in; | text-indent:-.25in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold;} | |||
font-weight:bold | |||
p.TableCells, li.TableCells, div.TableCells | p.TableCells, li.TableCells, div.TableCells | ||
{mso-style-name:"Table Cells"; | {mso-style-name:"Table Cells"; | ||
margin:0in; | margin:0in; | ||
margin-bottom:.0001pt; | margin-bottom:.0001pt; | ||
font-size:12.0pt; | font-size:12.0pt; | ||
font-family:"Times","serif";} | |||
font-family:"Times","serif" | |||
p.TableHeading, li.TableHeading, div.TableHeading | p.TableHeading, li.TableHeading, div.TableHeading | ||
{mso-style-name:"Table Heading"; | {mso-style-name:"Table Heading"; | ||
margin-top:4.0pt; | margin-top:4.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:4.0pt; | margin-bottom:4.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
page-break-after:avoid; | page-break-after:avoid; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif"; | font-family:"Arial","sans-serif"; | ||
font-weight:bold;} | |||
font-weight:bold | |||
p.TableBody, li.TableBody, div.TableBody | p.TableBody, li.TableBody, div.TableBody | ||
{mso-style-name:"Table Body"; | {mso-style-name:"Table Body"; | ||
margin-top:2.0pt; | margin-top:2.0pt; | ||
margin-right:0in; | margin-right:0in; | ||
margin-bottom:2.0pt; | margin-bottom:2.0pt; | ||
margin-left:0in; | margin-left:0in; | ||
font-size:10.0pt; | font-size:10.0pt; | ||
font-family:"Arial","sans-serif";} | |||
font-family:"Arial","sans-serif | |||
span.SubtitleChar | span.SubtitleChar | ||
{mso-style-name:"Subtitle Char"; | {mso-style-name:"Subtitle Char"; | ||
mso-style-link:Subtitle; | mso-style-link:Subtitle; | ||
font-family:"Cambria","serif";} | |||
font-family:"Cambria","serif | |||
/* Page Definitions */ | /* Page Definitions */ | ||
@page Section1 | |||
@page Section1 | |||
{size:8.5in 11.0in; | {size:8.5in 11.0in; | ||
margin:.5in .5in .5in .5in | margin:.5in .5in .5in .5in;} | ||
div.Section1 | div.Section1 | ||
{page:Section1;} | {page:Section1;} | ||
/* List Definitions */ | /* List Definitions */ | ||
ol | |||
ol | |||
{margin-bottom:0in;} | {margin-bottom:0in;} | ||
ul | ul | ||
| Line 1,266: | Line 215: | ||
--> | --> | ||
</style> | </style> | ||
</head> | </head> | ||
<body lang=EN-US link=blue vlink=purple | <body lang=EN-US link=blue vlink=purple> | ||
<div class=Section1> | <div class=Section1> | ||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | ||
style='margin-left:5.4pt;border-collapse:collapse;border:none | style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | ||
border:none'> | |||
<tr | <tr> | ||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | ||
padding:0in 5.4pt 0in 5.4pt'> | padding:0in 5.4pt 0in 5.4pt'> | ||
<p class=MsoTitle style='margin-right:26.1pt'>Pipetting & Making | <p class=MsoTitle style='margin-right:26.1pt'>Pipetting & Making | ||
Solutions</p> | Solutions</p> | ||
</td> | </td> | ||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | ||
padding:0in 5.4pt 0in 5.4pt'> | padding:0in 5.4pt 0in 5.4pt'> | ||
<p class=ExperimentNumber>Pg. 6</p> | <p class=ExperimentNumber>Pg. 6</p> | ||
| Line 1,331: | Line 238: | ||
</table> | </table> | ||
<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in | <p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
class=MsoIntenseEmphasis><span style=' | font-style:normal'><span style='font:7.0pt "Times New Roman"'> | ||
</span>I.<span style='font:7.0pt "Times New Roman"'> | </span>I.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span | </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Pipetting | font-weight:normal;font-style:normal'>Pipetting</span></u></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Learned to use pipettes properly</span></span></p> | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Learned to use | |||
pipettes properly | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Make sure to wipe down pipettes between | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Make sure to wipe | solutions (with Ethanol)</span></span></p> | ||
down pipettes between solutions (with Ethanol) | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Change tips every time you go between | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Change tips every | solutions</span></span></p> | ||
time you go between solutions | |||
<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in | <p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
class=MsoIntenseEmphasis><span style=' | font-style:normal'><span style='font:7.0pt "Times New Roman"'> | ||
</span>II.<span style='font:7.0pt "Times New Roman"'> | </span>II.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Solutions</span></u></span></p> | |||
< | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Task: Make 0.3M KCl</span></span></p> | |||
<p class=MsoNormal style='margin-left:1. | <p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
style=' | </span>i.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | ||
</span></span></span | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | font-style:normal'>Salt (KCl): CAS 7447-40-7</span></span></p> | ||
KCl | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in | <p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
class=MsoIntenseEmphasis><span style=' | font-style:normal'><span style='font:7.0pt "Times New Roman"'> | ||
</span>ii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
</span> | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'> | font-style:normal'>MW: 74.56g/mol</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in | <p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal | font-style:normal'><span style='font:7.0pt "Times New Roman"'> | ||
</span>iii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
</span>iii.<span style='font:7.0pt "Times New Roman"'> </span></span></span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>2.2368 g / 100mL | font-style:normal'>2.2368 g / 100mL</span></span></p> | ||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | <p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | ||
font-weight:normal;font-style:normal' | font-weight:normal;font-style:normal'> </span></span></p> | ||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | ||
style='margin-left:5.4pt;border-collapse:collapse;border:none | style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | ||
border:none'> | |||
<tr | <tr> | ||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | <td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | ||
padding:0in 5.4pt 0in 5.4pt'> | padding:0in 5.4pt 0in 5.4pt'> | ||
<p class=MsoTitle style='margin-right:26.1pt'>Plate Creation & Cell | <p class=MsoTitle style='margin-right:26.1pt'>Plate Creation & Cell | ||
Culture</p> | Culture</p> | ||
</td> | </td> | ||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | <td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | ||
padding:0in 5.4pt 0in 5.4pt'> | padding:0in 5.4pt 0in 5.4pt'> | ||
<p class=ExperimentNumber>Pg. 7</p> | <p class=ExperimentNumber>Pg. 7</p> | ||
| Line 1,431: | Line 318: | ||
</table> | </table> | ||
<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in | <p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
class=MsoIntenseEmphasis><span style=' | font-style:normal'><span style='font:7.0pt "Times New Roman"'> | ||
</span>I.<span style='font:7.0pt "Times New Roman"'> | </span>I.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span | </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Steps for | font-weight:normal;font-style:normal'>Steps for creating a plate</span></u></span><span | ||
creating a plate</span></u></span><span class=MsoIntenseEmphasis><span | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
style='color:windowtext;font-weight:normal;font-style:normal'>: | font-style:normal'>:</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Dissolve 25g LB in 800mL of H<sub>2</sub>O</span></span></p> | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Dissolve 25g LB | |||
in 800mL of H<sub>2</sub>O | |||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | <p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Bring the final | style='color:windowtext;font-weight:normal;font-style:normal'>Bring the final | ||
volume to 1L in a 2L flask | volume to 1L in a 2L flask</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | <p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Add 15g | style='color:windowtext;font-weight:normal;font-style:normal'>Add 15g | ||
Bactoagar, cover with foil, autoclave for 20-30 minutes | Bactoagar, cover with foil, autoclave for 20-30 minutes</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Bring back to lab, cool to 50</span></span><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Bring back to | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
lab, cool to 50</span></span><span class=MsoIntenseEmphasis><span | |||
style=' | |||
font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>C </span></span><span | style='color:windowtext;font-weight:normal;font-style:normal'>C </span></span><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>while | |||
stirring</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'> | |||
class=MsoIntenseEmphasis | </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | ||
font-style:normal'> | font-weight:normal;font-style:normal'>Add100mg AMP & let swirl until | ||
dissolved</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>d.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Pour it into the plates (about 2-3mm | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | deep)</span></span></p> | ||
<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
<p class=MsoNormal style='margin-left:.75in;text-indent:-.75in | |||
class=MsoIntenseEmphasis><span style=' | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | </span>II.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span | </span></span></span><span class=MsoIntenseEmphasis><u><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Cell Culture</span></u></span><span | font-weight:normal;font-style:normal'>Cell Culture</span></u></span><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>: | font-style:normal'>:</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Use solution of LB with AMP</span></span></p> | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Use solution of | |||
LB with AMP | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | <p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>Ensure to keep the bags sealed to keep everything sterile | font-style:normal'>Ensure to keep the bags sealed to keep everything sterile </span></span></p> | ||
<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | <p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>*Note once a bag of plates has been opened, the whole thing | font-style:normal'>*Note once a bag of plates has been opened, the whole thing | ||
must be used | must be used</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | <p class=MsoNormal style='margin-left:1.0in;text-indent:.25in'><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>The tubes allow the solution to be aerated | font-style:normal'>The tubes allow the solution to be aerated</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | <p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight | style='color:windowtext;font-weight:normal'>The tubes with bacteria should look | ||
tubes with bacteria should look cloudy upon visual inspection—can be confirmed | cloudy upon visual inspection—can be confirmed with cell density measurement | ||
with cell density measurement (using the Spec) | (using the Spec)</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>b.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Sterilize the autopipette, set to 1mL | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Sterilize the | (make a 2mL culture, however)</span></span></p> | ||
autopipette, set to 1mL (make a 2mL culture, however) | |||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | <p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Add 2mL of the LB | style='color:windowtext;font-weight:normal;font-style:normal'>Add 2mL of the LB | ||
(for a miniprep); you will have to add 1mL, two times | (for a miniprep); you will have to add 1mL, two times</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'>c.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
</span></span></span | font-weight:normal;font-style:normal'>Label one as the control and another as | ||
style='color:windowtext;font-weight:normal;font-style:normal'>Label one as the | your experimental sample</span></span></p> | ||
control and another as your experimental sample | |||
<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span | <p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span | ||
style='color:windowtext;font-weight:normal;font-style:normal'>*Label the tubes | style='color:windowtext;font-weight:normal;font-style:normal'>*Label the tubes | ||
with your Initials, Sample Name (control or experimental) and the date< | with your Initials, Sample Name (control or experimental) and the date</span></span></p> | ||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>d.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Dip the culture scraper in ethanol and | |||
flame lightly (2x) to sterilize</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>e.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Let the scraper cool for a few seconds</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>f.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Scrape off a tiny bit of bacteria off of | |||
the plate and place the scraper into the tube and shake around to ensure the | |||
bacteria breaks off into the tube</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>g.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Set incubator at 37</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>C overnight and | |||
let shake gently for 12-14hrs</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> </span></span></p> | |||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | |||
style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | |||
border:none'> | |||
<tr> | |||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoTitle style='margin-right:26.1pt'>Transformation of E.coli with | |||
Recombinant DNA</p> | |||
</td> | |||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=ExperimentNumber>Pg. 9</p> | |||
</td> | |||
</tr> | |||
</table> | |||
<p class=MsoNormal style='margin-left:.75in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>*note, </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>the | |||
heat shock</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> to the tubes is very important for | |||
transcription to occur</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> <u>+ Controls</u>:</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_I13522 : | |||
pTetGFP</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_J45220</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> <u>Group 1:</u></span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_R0011</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_J45219</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> <u>Group 2:</u></span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_I14032 / | |||
BBa_R0051 (same)</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_J06702</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> <u>Group 3:</u></span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_J04500</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> BBa_J04630 / | |||
BBa_I13401</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='text-indent:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Rima & I did the transformation | |||
for Group 2:</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>I.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>BBa_I14032</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name: | |||
</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>promoter P(Lac) IQ</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Sequence: | |||
</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>promoter</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'> </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>(-35, -10 bases before coding)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>: 2007, Plate 2, Well 17K</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>d.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid: | |||
</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>pSB2K3</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>BBa_R0051</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name:</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> promoter (lamda cl regulated)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Sequence:</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> R0051 promoter</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location: | |||
</span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>2007, Plate 1, Well 9C</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><i>d.<span | |||
style='font:7.0pt "Times New Roman"'> </span></i><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid:</span></span><i> | |||
</i>pSB1A2</p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>III.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>BBa_J06702</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Name:</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> mCherry, bacterial with RBS & forward terminator</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>Sequence</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>: </span></span></p> | |||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | |||
style='margin-left:159.5pt;border-collapse:collapse;border:none'> | |||
<tr> | |||
<td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'>RBS</span></span></p> | |||
</td> | |||
<td width=85 valign=top style='width:64.05pt;border:solid windowtext 1.0pt; | |||
border-left:none;padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'>mCherry</span></span></p> | |||
</td> | |||
<td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt; | |||
border-left:none;padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'>Terminator</span></span></p> | |||
</td> | |||
<td width=101 valign=top style='width:76.05pt;border:solid windowtext 1.0pt; | |||
border-left:none;padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'>Terminator</span></span></p> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td width=70 valign=top style='width:52.8pt;border:solid windowtext 1.0pt; | |||
border-top:none;padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>B0034</span></span></p> | |||
</td> | |||
<td width=85 valign=top style='width:64.05pt;border-top:none;border-left: | |||
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>J06504</span></span></p> | |||
</td> | |||
<td width=101 valign=top style='width:76.05pt;border-top:none;border-left: | |||
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>B0010</span></span></p> | |||
</td> | |||
<td width=101 valign=top style='width:76.05pt;border-top:none;border-left: | |||
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>B0012</span></span></p> | |||
</td> | |||
</tr> | |||
</table> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Location:</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> 2007, Plate 2, Well 11D</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>d.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>Plasmid:</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> pSB1A2</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>IV.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Cells Used: E.coli: JM109 (Promega); | |||
Catalog #: L2001</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>V.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>3 Plates Made:</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>a.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>Control: 100μL LB / Cell</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>b.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>Test Plate 1: 100μL LB/ Cell</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>c.<span | |||
style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>Test Plate 2: 1μL cell : 99μ H<sub>2</sub>O</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Actual Transformation Procedure:</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>I.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>If using DNA from the registry, dilute | |||
with 9μL of H<sub>2</sub>O. If using our own DNA, use 200ng. </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>inject the water directly into the well, | |||
titerate, and then pipette it out</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Thaw 100μL competent cells on ice. | |||
(50</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>μL with our own/ 100</span></span> <span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μ if commercial cells used)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>III.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Mix competent cells with 50</span></span> | |||
<span class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μ CaCl<sub>2</sub> (only if using our own cells)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>IV.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add 4</span></span> <span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μL of registry DNA, or 200ng of our own DNA to the | |||
cells. </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>V.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Put in the ice bath for about 20 minutes. | |||
</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VI.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Remove and immediately put in the hot | |||
water bath (42</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>°</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>C) to heat shock for 45-60 sec.</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VII.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Place back in the ice bath for 2 minutes. | |||
</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
style=' | </span>VIII.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span | </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | font-weight:normal;font-style:normal'>Add 900 μL ice cold, antibiotic free | ||
LB to the cells</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
style=' | </span>IX.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span></span>< | </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | font-weight:normal;font-style:normal'>Incubate at 37</span></span><span | ||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>C for 90 minutes | |||
in the shaking incubator.</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
style=' | </span>X.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span | </span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | ||
style='color:windowtext;font-weight:normal;font-style:normal'> | font-weight:normal;font-style:normal'>Dilute as desired, make the necessary | ||
controls, and plate cells using 100 μL / plate. (200 if not commercial)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-.25in | <p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | ||
class=MsoIntenseEmphasis><span style=' | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
style=' | </span>XI.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Incubate upside down (so that the label | |||
you wrote on it is face up) at 37</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>°</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>C for 18-24 hours, then refrigerate. </span></span></p> | |||
< | <p class=MsoNormal style='margin-left:1.5in;text-indent:-9.0pt'><span | ||
style=' | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | ||
font-style:normal'> | font-style:normal'>a.<span style='font:7.0pt "Times New Roman"'> | ||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>The controls should not have bacteria | |||
growing on them!</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | |||
style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | |||
border:none'> | |||
<tr> | |||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoTitle style='margin-right:26.1pt'>Miniprep</p> | |||
</td> | |||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=ExperimentNumber>Pg. 12</p> | |||
</td> | |||
</tr> | |||
</table> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>*Start with | |||
Parts: E0241 (x3), R0010, R0040, C0040 from the registry*</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>E0241: PoPs to GFP Converter</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; | |||
font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>RBS: B0032</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; | |||
font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>GFP: E0040</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; | |||
font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Terminator: B0016</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>R0010: Promoter Lac I (Regulator) </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>R0040: Promoter (tetR, negative)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>C0040: Tetracycline repressor from | |||
transposon Tn10 (+LVA) [note that +LVA is a degradation tag]</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Look out for:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'> </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>Control should not be cloudy at all, culture should have gas | |||
bubbles, should be cloudy, especially at the bottom</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> *If | |||
you start seeing sediment at the bottom, the cells </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>may</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> have lysed and let proteins into the tube*</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='text-indent:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Procedure (General):</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>Use the QIAGEN | |||
Kit to separate the plasmid DNA (so it should not have much chromosomal DNA)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>I.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Grow cells for 12 to 14 hours</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Suspend cells in </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>i.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>buffer 1 (with glucose & EDTA)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>ii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>buffer 2 (SDS-detergent, NaOH) [SDS gets rid of the membrane | |||
and NaOH denatures the plasmid and DNA]</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.75in;text-indent:-1.75in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>iii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>buffer 3 (K Acetate / Acetic Acid)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>-things become an insoluble precipitate</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>-renature the DNA/plasmid</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>-only the plasmid DNA will renature properly</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>-the plasmid DNA remains soluble & the chromosomal DNA | |||
will become the precipitate</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>III.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Columns: use EtOH to extract DNA</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Procedure (Specific):</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> *make | |||
sure to check your buffers for salt precipitation—this may indicate a change in | |||
concentration*</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>I.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Get solution and put 1.5mL into an Eppendorf | |||
tube to centrifuge </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>-after | |||
centrifuging for 30 seconds @ 13k RPM, remove the supernatant</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>-add more | |||
solution, and centrifuge again using same procedure</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add 250</span></span> <span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μL PI to tube after the supernatant is </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>fully | |||
removed</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>III.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add 250 μL P2, and invert 4 to 6 | |||
times</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>-immediately add | |||
350</span></span> <span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>μL N3, and invert again 4 to 6 times</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>IV.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Dispose of the flow through, add 0.5mL of | |||
buffer PB (that is </span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>milliliter not microliter!)</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'> then centrifuge for 30-60 seconds</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>V.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add 750</span></span> <span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μL PE, then centrifuge again, remove the waste, | |||
centrifuge again</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VI.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add 50</span></span> <span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>μL BE buffer</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VII.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Measure the DNA concentration using | |||
Nanodrop</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.25in;text-indent:-1.25in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VIII.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Ethanol Precipitation </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal'>(see | |||
separate procedure)</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal'> </span></span></p> | |||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | |||
style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | |||
border:none'> | |||
<tr> | |||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoTitle style='margin-right:26.1pt'>Ethanol Precipitation (for | |||
>200bp)</p> | |||
</td> | |||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=ExperimentNumber>Pg. 15</p> | |||
</td> | |||
</tr> | |||
</table> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> *used to purify the DNA | |||
(really small fragments) or to increase the concentration of it</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>I.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Add:</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>i.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>3 volumes of COLD 100% EtOH</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>ii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>1/10 volume of 3M sodium acetate</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-1.5in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>iii.<span style='font:7.0pt "Times New Roman"'> </span></span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>1 μL GlycoBlue</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>II.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Incubate for 1 hr @ 80</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>C</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>III.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Centrifuge for 30min @ 0</span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>°</span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>C</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>IV.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Remove supernatant</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>V.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Air Dry</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-1.0in'><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'><span style='font:7.0pt "Times New Roman"'> | |||
</span>VI.<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Resuspend in H<sub>2</sub>O</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Specific Procedure Example:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> For | |||
50 μL:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> Add | |||
150 μL EtOH</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 20 | |||
μL NaAc.</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> For | |||
100 μL:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> Add 300 | |||
μL EtOH</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 40 | |||
μL NaAc.</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 | |||
style='margin-left:5.4pt;background:#CCCCCC;border-collapse:collapse; | |||
border:none'> | |||
<tr> | |||
<td width=488 style='width:365.7pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=MsoTitle style='margin-right:26.1pt'>Gel Electrophoresis</p> | |||
</td> | |||
<td width=136 style='width:102.3pt;border:none;border-bottom:solid windowtext 2.25pt; | |||
padding:0in 5.4pt 0in 5.4pt'> | |||
<p class=ExperimentNumber>Pg. 16</p> | |||
</td> | |||
</tr> | |||
</table> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> *applying an electric field | |||
over the gel will separate fragments by size</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> *compare the movement of the | |||
DNA to the ladder</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span><span | |||
class=MsoIntenseEmphasis><u><span style='color:windowtext;font-weight:normal'>*ensure | |||
that the well is on the negative side!*</span></u></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>DNA has a (-) charge because of the | |||
phosphate groups</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>moves toward the anode (+) and away from | |||
the cathode (-)</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>DNA is separated by size, distance ~ 1 / | |||
molecular weight</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.5in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Wingdings;color:windowtext; | |||
font-weight:normal;font-style:normal'>§<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>100bp-50kbp</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>Gel acts as a sieve: 0.3% large fragments | |||
to 2% small fragments</span></span></p> | |||
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><span | |||
class=MsoIntenseEmphasis><span style='font-family:Symbol;color:windowtext; | |||
font-weight:normal;font-style:normal'>®<span style='font:7.0pt "Times New Roman"'> | |||
</span></span></span><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'>after the gel is made, put it in the | |||
microwave for 1minute, then </span></span><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>cool the bottle in H<sub>2</sub>O, | |||
and take the lid off</span></span></p> | |||
<p class=MsoNormal><span class=MsoIntenseEmphasis><span style='color:windowtext; | |||
font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Casting Gel:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 1% | |||
Agarose, 0.5g Agarose</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 50mL | |||
TBE + SYBR Safe</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>Dilute the 5xTBE | |||
to 0.5x</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'>Add 2 μL of | |||
the loading dye!</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> </span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'>Ladder Formula:</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal'> </span></span><span | |||
class=MsoIntenseEmphasis><span style='color:windowtext;font-weight:normal; | |||
font-style:normal'>1 μL 1kbase ladder</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 2 μL | |||
BlueJuice Dye</span></span></p> | |||
<p class=MsoNormal style='margin-left:.5in'><span class=MsoIntenseEmphasis><span | |||
style='color:windowtext;font-weight:normal;font-style:normal'> 7 μL | |||
H<sub>2</sub>O</span></span></p> | |||
</div> | </div> | ||
| Line 1,592: | Line 1,343: | ||
</html> | </html> | ||
Latest revision as of 19:44, 11 March 2008
<html>
<head> <meta http-equiv=Content-Type content="text/html; charset=windows-1252"> <meta name=Generator content="Microsoft Word 12 (filtered)"> <title>This is an experiment title</title> <style> </style>
</head>
<body lang=EN-US link=blue vlink=purple>
|
Pipetting & Making Solutions |
Pg. 6 |
I. Pipetting
a. Learned to use pipettes properly
b. Make sure to wipe down pipettes between solutions (with Ethanol)
c. Change tips every time you go between solutions
II. Solutions
a. Task: Make 0.3M KCl
i. Salt (KCl): CAS 7447-40-7
ii. MW: 74.56g/mol
iii. 2.2368 g / 100mL
|
Plate Creation & Cell Culture |
Pg. 7 |
I. Steps for creating a plate:
a. Dissolve 25g LB in 800mL of H2O
Bring the final volume to 1L in a 2L flask
Add 15g Bactoagar, cover with foil, autoclave for 20-30 minutes
b. Bring back to lab, cool to 50°C while stirring
c. Add100mg AMP & let swirl until dissolved
d. Pour it into the plates (about 2-3mm deep)
II. Cell Culture:
a. Use solution of LB with AMP
Ensure to keep the bags sealed to keep everything sterile
*Note once a bag of plates has been opened, the whole thing must be used
The tubes allow the solution to be aerated
The tubes with bacteria should look cloudy upon visual inspection—can be confirmed with cell density measurement (using the Spec)
b. Sterilize the autopipette, set to 1mL (make a 2mL culture, however)
Add 2mL of the LB (for a miniprep); you will have to add 1mL, two times
c. Label one as the control and another as your experimental sample
*Label the tubes with your Initials, Sample Name (control or experimental) and the date
d. Dip the culture scraper in ethanol and flame lightly (2x) to sterilize
e. Let the scraper cool for a few seconds
f. Scrape off a tiny bit of bacteria off of the plate and place the scraper into the tube and shake around to ensure the bacteria breaks off into the tube
g. Set incubator at 37°C overnight and let shake gently for 12-14hrs
|
Transformation of E.coli with Recombinant DNA |
Pg. 9 |
*note, the heat shock to the tubes is very important for transcription to occur
+ Controls:
BBa_I13522 : pTetGFP
BBa_J45220
Group 1:
BBa_R0011
BBa_J45219
Group 2:
BBa_I14032 / BBa_R0051 (same)
BBa_J06702
Group 3:
BBa_J04500
BBa_J04630 / BBa_I13401
Rima & I did the transformation for Group 2:
I. BBa_I14032
a. Name: promoter P(Lac) IQ
b. Sequence: promoter (-35, -10 bases before coding)
c. Location: 2007, Plate 2, Well 17K
d. Plasmid: pSB2K3
II. BBa_R0051
a. Name: promoter (lamda cl regulated)
b. Sequence: R0051 promoter
c. Location: 2007, Plate 1, Well 9C
d. Plasmid: pSB1A2
III. BBa_J06702
a. Name: mCherry, bacterial with RBS & forward terminator
b. Sequence:
|
RBS |
mCherry |
Terminator |
Terminator |
|
B0034 |
J06504 |
B0010 |
B0012 |
c. Location: 2007, Plate 2, Well 11D
d. Plasmid: pSB1A2
IV. Cells Used: E.coli: JM109 (Promega); Catalog #: L2001
V. 3 Plates Made:
a. Control: 100μL LB / Cell
b. Test Plate 1: 100μL LB/ Cell
c. Test Plate 2: 1μL cell : 99μ H2O
Actual Transformation Procedure:
I. If using DNA from the registry, dilute with 9μL of H2O. If using our own DNA, use 200ng.
a. inject the water directly into the well, titerate, and then pipette it out
II. Thaw 100μL competent cells on ice. (50 μL with our own/ 100 μ if commercial cells used)
III. Mix competent cells with 50 μ CaCl2 (only if using our own cells)
IV. Add 4 μL of registry DNA, or 200ng of our own DNA to the cells.
V. Put in the ice bath for about 20 minutes.
VI. Remove and immediately put in the hot water bath (42°C) to heat shock for 45-60 sec.
VII. Place back in the ice bath for 2 minutes.
VIII. Add 900 μL ice cold, antibiotic free LB to the cells
IX. Incubate at 37°C for 90 minutes in the shaking incubator.
X. Dilute as desired, make the necessary controls, and plate cells using 100 μL / plate. (200 if not commercial)
XI. Incubate upside down (so that the label you wrote on it is face up) at 37°C for 18-24 hours, then refrigerate.
a. The controls should not have bacteria growing on them!
|
Miniprep |
Pg. 12 |
*Start with Parts: E0241 (x3), R0010, R0040, C0040 from the registry*
® E0241: PoPs to GFP Converter
§ RBS: B0032
§ GFP: E0040
§ Terminator: B0016
® R0010: Promoter Lac I (Regulator)
® R0040: Promoter (tetR, negative)
® C0040: Tetracycline repressor from transposon Tn10 (+LVA) [note that +LVA is a degradation tag]
Look out for:
Control should not be cloudy at all, culture should have gas bubbles, should be cloudy, especially at the bottom
*If you start seeing sediment at the bottom, the cells may have lysed and let proteins into the tube*
Procedure (General):
Use the QIAGEN Kit to separate the plasmid DNA (so it should not have much chromosomal DNA)
I. Grow cells for 12 to 14 hours
II. Suspend cells in
i. buffer 1 (with glucose & EDTA)
ii. buffer 2 (SDS-detergent, NaOH) [SDS gets rid of the membrane and NaOH denatures the plasmid and DNA]
iii. buffer 3 (K Acetate / Acetic Acid)
-things become an insoluble precipitate
-renature the DNA/plasmid
-only the plasmid DNA will renature properly
-the plasmid DNA remains soluble & the chromosomal DNA will become the precipitate
III. Columns: use EtOH to extract DNA
Procedure (Specific):
*make sure to check your buffers for salt precipitation—this may indicate a change in concentration*
I. Get solution and put 1.5mL into an Eppendorf tube to centrifuge
-after centrifuging for 30 seconds @ 13k RPM, remove the supernatant
-add more solution, and centrifuge again using same procedure
II. Add 250 μL PI to tube after the supernatant is fully removed
III. Add 250 μL P2, and invert 4 to 6 times
-immediately add 350 μL N3, and invert again 4 to 6 times
IV. Dispose of the flow through, add 0.5mL of buffer PB (that is milliliter not microliter!) then centrifuge for 30-60 seconds
V. Add 750 μL PE, then centrifuge again, remove the waste, centrifuge again
VI. Add 50 μL BE buffer
VII. Measure the DNA concentration using Nanodrop
VIII. Ethanol Precipitation (see separate procedure)
|
Ethanol Precipitation (for >200bp) |
Pg. 15 |
*used to purify the DNA (really small fragments) or to increase the concentration of it
I. Add:
i. 3 volumes of COLD 100% EtOH
ii. 1/10 volume of 3M sodium acetate
iii. 1 μL GlycoBlue
II. Incubate for 1 hr @ 80°C
III. Centrifuge for 30min @ 0°C
IV. Remove supernatant
V. Air Dry
VI. Resuspend in H2O
Specific Procedure Example:
For 50 μL:
Add 150 μL EtOH
20 μL NaAc.
For 100 μL:
Add 300 μL EtOH
40 μL NaAc.
|
Gel Electrophoresis |
Pg. 16 |
*applying an electric field over the gel will separate fragments by size
*compare the movement of the DNA to the ladder
*ensure that the well is on the negative side!*
® DNA has a (-) charge because of the phosphate groups
® moves toward the anode (+) and away from the cathode (-)
® DNA is separated by size, distance ~ 1 / molecular weight
§ 100bp-50kbp
® Gel acts as a sieve: 0.3% large fragments to 2% small fragments
® after the gel is made, put it in the microwave for 1minute, then cool the bottle in H2O, and take the lid off
Casting Gel:
1% Agarose, 0.5g Agarose
50mL TBE + SYBR Safe
Dilute the 5xTBE to 0.5x
Add 2 μL of the loading dye!
Ladder Formula:
1 μL 1kbase ladder
2 μL BlueJuice Dye
7 μL H2O
</body>
</html>
