http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Gcxmatt&year=&month=2010.igem.org - User contributions [en]2024-03-29T00:15:49ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/File:WCAS.jpgFile:WCAS.jpg2011-02-16T16:40:36Z<p>Gcxmatt: </p>
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<div></div>Gcxmatthttp://2010.igem.org/Team:NorthwesternTeam:Northwestern2011-02-16T16:39:44Z<p>Gcxmatt: </p>
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
[[Image:ohgodtimithebacteriaareangry.jpg|936px|center]]<br />
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|style="padding: 5" valign="top" width="60%"|<br />
<br />
{|cellspacing="30" border="0"<br />
|'''SCIN - Self-regenerating Chitin INduction'''<br />
<br />
Chitin, found in the exoskeletons of insects and crustaceans as well as the cell walls of fungi, is one of the most abundant organic polymers in nature. Like keratin in skin, it comprises the protective outer layer of these organisms. Our goal is to generate a layer of chitin from a lawn of bacteria (Escherichia coli) in response to an external molecular cue. This cue induces chitin synthesis (fast) and cell lysis (slow), allowing for a build-up of chitin followed by cell lysis and subsequent release into the top layer of the lawn. Abrasions expose cells to the external cue for self-repair. This would create a regenerative chitin biolayer with potential medical and industrial applications.<br />
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{|<br />
|align="center"|To find out more, click the links below:<br />
<br />
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<table align="center"><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Northwestern/Project"><img width="220" class="icon" src="https://static.igem.org/mediawiki/2010/e/ee/Projectoverview_copy.jpg"></a></td><br />
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<table align="center"><br />
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<td><a href="https://2010.igem.org/Team:Northwestern/Project/Modeling"><img width="65" class="icon" src="https://static.igem.org/mediawiki/2010/a/a7/NU_modeling.jpg"></a></td><br />
<td><a href="https://2010.igem.org/Team:Northwestern/Project/Chassis"><img width="65" class="icon" src="https://static.igem.org/mediawiki/2010/a/a6/Nuchassis.jpg"></a></td><br />
<td><a href="https://2010.igem.org/Team:Northwestern/Project/Lac"><img width="65" class="icon" src="https://static.igem.org/mediawiki/2010/1/18/Nulac.jpg"></a></td><br />
<td><a href="https://2010.igem.org/Team:Northwestern/Project/Chitin Synthesis"><img width="65" class="icon" src="https://static.igem.org/mediawiki/2010/4/4e/Nuchitin.jpg"></a></td><br />
<td><a href="https://2010.igem.org/Team:Northwestern/Project/Apoptosis"><img width="65" class="icon" src="https://static.igem.org/mediawiki/2010/c/cb/Nuapop.jpg"></a></td><br />
</tr><br />
<tr><br />
<td align="center">Modeling</td><br />
<td align="center">Chassis</td><br />
<td align="center">Induction</td><br />
<td align="center">Chitin</td><br />
<td align="center">Apoptosis</td><br />
</tr><br />
</table><br />
</html><br />
|}<br />
|[[Image:PosterChild.jpg|330px|center]]<br />
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</a><br />
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<br />
|-<br />
|colspan="3"|<br />
<br />
== '''Northwestern University''' ==<br />
Northwestern University in Evanston, IL, combines innovative teaching and pioneering research in a highly collaborative environment that transcends traditional academic boundaries.<br />
<br />
Our lab facilities are located at: Technological Institute 2145 Sheridan Road, Evanston, IL 60208<br />
<br />
Find out more about [http://www.northwestern.edu Northwestern University]<br />
<br />
Find out more about [http://www.mccormick.northwestern.edu/ McCormick School of Engineering]<br />
<br />
Find out more about [http://www.weinberg.northwestern.edu/ Weinberg College of Arts and Sciences]<br />
<br />
=='''Sponsors'''==<br />
<br />
[[image:NU.jpg|200px]] [[image:NEB.jpg|200px]] [[image:Promega.jpg|200px]] [[image:invitrogen.jpg|200px]] [[image:VWR.jpg|200px]] [[image:Picture1.jpg|200px]] [[image:BIF.jpg|200px]] [[image:EPOCH.jpg|200px]]<br />
[[image:tit.jpg|400px]] [[image:WCAS.jpg|200px]]<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:17:09Z<p>Gcxmatt: /* Notebook */</p>
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
='''Notebook'''=<br />
<br />
[[Brainstorming April-June 2010]]<br />
<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:16:58Z<p>Gcxmatt: /* Notebook */</p>
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
='''Notebook'''=<br />
<br />
[[Brainstorming April-June 2010]]<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:16:48Z<p>Gcxmatt: /* April */</p>
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<div>__NOTOC__<br />
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</style><br />
</html><br />
<br />
<html><br />
<head><br />
<style><br />
body {<br />
background: #EFCDF8; <!-- EEDCC8 url(https://static.igem.org/mediawiki/2010/3/38/BG1.jpg) repeat-y; --><br />
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</style><br />
</head><br />
</html><br />
<br />
[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
='''Notebook'''=<br />
<br />
<br />
[[Brainstorming April-June 2010]]<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:16:29Z<p>Gcxmatt: /* Notebook */</p>
<hr />
<div>__NOTOC__<br />
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#globalWrapper {<br />
background-color: transparent;<br />
padding-bottom:0px;<br />
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<br />
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#content {<br />
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</style><br />
</html><br />
<br />
<html><br />
<head><br />
<style><br />
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</style><br />
</head><br />
</html><br />
<br />
[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
='''Notebook'''=<br />
<br />
<br />
=='''April'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:16:06Z<p>Gcxmatt: /* Notebook */</p>
<hr />
<div>__NOTOC__<br />
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</style><br />
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<br />
<html><br />
<head><br />
<style><br />
body {<br />
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</head><br />
</html><br />
<br />
[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
='''Notebook'''=<br />
<br />
=='''April'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:15:51Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
<br />
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<html><br />
<style><br />
/* Wiki Hacks - START */<br />
#globalWrapper {<br />
background-color: transparent;<br />
padding-bottom:0px;<br />
border: none;<br />
}<br />
#top-section {<br />
height: 0px;<br />
margin-top: 0px;<br />
margin-left: auto;<br />
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right: 40px;<br />
} <br />
<br />
<br />
/* Wiki Hacks - END */<br />
<br />
<br />
#content {<br />
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float: center;<br />
border: 0px;<br />
}<br />
<br />
#menubar {<br />
background-color:transparent;<br />
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<br />
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/* Wiki Hacks - END */<br />
</style><br />
</html><br />
<br />
<html><br />
<head><br />
<style><br />
body {<br />
background: #EFCDF8; <!-- EEDCC8 url(https://static.igem.org/mediawiki/2010/3/38/BG1.jpg) repeat-y; --><br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
=='''Notebook'''==<br />
<br />
=='''April'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T02:13:49Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
<br />
<br />
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}<br />
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</head><br />
</html><br />
<br />
[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
=='''Notebook'''==<br />
<br />
=='''April'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
----<br />
<br />
=='''June'''==<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
----<br />
<br />
=='''July'''==<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
----<br />
<br />
=='''August'''==<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
----<br />
<br />
=='''September'''==<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
----<br />
<br />
=='''October'''==<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
----<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Week15_9/19/10-9/25/10Week15 9/19/10-9/25/102010-10-27T02:11:53Z<p>Gcxmatt: /* 9/25/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week15 Highlights===<br />
Classes at Northwestern started this week. This complicated our functionality as a team due to scheduling. Thus, we paired up and began working on different portions of the project on our own.<br />
<br />
*Timi/Kevin: CP-LacPI assembly<br />
<br />
*Matt/Sean: Chitin synthase<br />
<br />
*Ben: Site directed mutagenesis<br />
<br />
*Ragan: MAGE<br />
<br />
We submitted our iGEM abstract and has a group meeting for the first time since everyone left to go back home for a few weeks. We realized that something was wrong with our originally PCRed CHS3, so we remade it. However, we were only able to get 25-35 ng/ul of DNA. We also began an extra weekly meeting on Saturdays around 4pm to work on our poster, presentation, and wiki pages.<br />
<br />
----<br />
<br />
===9/20/10===<br />
iGEM abstracts were due tonight. We had an after-hours lab meeting to finish things up. Prof. Leonard stopped by to help us make some final edits and to figure out how much money we had left in the budget.<br />
<br />
We also took this time to catch eachother up on what we had been working on since team members started leaving for home.<br />
----<br />
<br />
===9/21/10===<br />
Timi and Kevin poured a large gel prior to heading home for the night.<br />
----<br />
<br />
===9/22/10===<br />
Timi and Kevin loaded digested CP-LacPI assemblies into gels this morning and Kevin ran the gel between classes.<br />
----<br />
<br />
===9/23/10===<br />
Early 8am team meeting with Prof. Mordacq.<br />
<br />
CHS3 made, low conc (25-35) 5 tubes of about 80ul each in 4degree<br />
----<br />
<br />
===9/24/10===<br />
Digested LacP-RBS(2) with ES.<br />
Digested Tet plasmid with ES.<br />
----<br />
===9/25/10===<br />
We had a group lab meeting to begin working on the poster and presentation.<br />
<br />
Timi and Kevin put 18 overnight cultures of CP-LacPI assemblies into the incubator with Tet antibiotic.<br />
----</div>Gcxmatthttp://2010.igem.org/Week14_9/12/10-9/18/10Week14 9/12/10-9/18/102010-10-27T02:11:29Z<p>Gcxmatt: /* 9/17/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week14 Highlights===<br />
We ran multiple gels to test our LacP-RBS and LacP-RBS-CHS3 assemblies. We continued preparing and assembling our combinations of Cp-LacPi-GFP for characterization. We also poured more Chlor and Tet plates using smaller (60mm x 15mm) petri dishes given to us by VWR.<br />
----<br />
===9/13/10===<br />
Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.<br />
----<br />
<br />
===9/14/10===<br />
Inoculated Apop1<br />
<br />
Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes<br />
<br />
Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.<br />
----<br />
<br />
===9/15/10===<br />
Miniprepped LacP-RBS-CHS3 and Apop1<br />
<br />
Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)<br />
<br />
Kevin put 15 overnight cultures into the 37 degree incubator at 6pm. <br />
<br />
Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate). <br />
<br />
Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.<br />
----<br />
<br />
===9/16/10===<br />
Ran digested LacP-RBS-CHS3 on a gel:<br />
*Majority contained 1 band around 3600bp<br />
*3 contained 3 bands (2000bp, 1600bp, 1100bp)<br />
<br />
Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.<br />
----<br />
<br />
===9/17/10===<br />
Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel<br />
LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp<br />
<br />
Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.<br />
<br />
Results: Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.<br />
<br />
CP3-LacPi2 retrans:<br />
<br />
1) 155.3<br />
<br />
2) 135.1<br />
<br />
3) 76.8<br />
<br />
CP1-LacPi2 seq:<br />
<br />
1) 102.9<br />
<br />
2) 91.7<br />
<br />
3) 71.7<br />
<br />
CP2-LacPi2:<br />
<br />
1) 83.8<br />
<br />
2) 89.6<br />
<br />
CP1-LacPi2<br />
<br />
1) 54.7<br />
<br />
2) 94.7<br />
<br />
CP1-LacPi1:<br />
<br />
1) 108.8<br />
<br />
2) 66.0<br />
<br />
3) 36.5<br />
<br />
The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.<br />
<br />
<br />
PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.<br />
<br />
Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.<br />
----</div>Gcxmatthttp://2010.igem.org/Week13_9/5/10-9/11/10Week13 9/5/10-9/11/102010-10-27T02:10:48Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week13 Highlights===<br />
We began reorganizing our lab space and equipment in preparation for a fall quarter class that would be utilizing the majority of the lab. We attempted standard assembly of LacP-RBS-CHS3 because the presence of a P site in CHS3 prevented 3A assembly. <br />
----<br />
===9/6/10===<br />
Kevin did some reorganizing to get the lab back in order for Prof. Mordacq's class this fall. He consolidated the bench space, the -20 refrigerator and the cold room.<br />
<br />
----<br />
<br />
===9/7/10===<br />
Day Off<br />
----<br />
<br />
===9/8/10===<br />
Day Off<br />
----<br />
===9/9/10===<br />
Assembled LacP-RBS1-CHS3 in a Chlor backbone plasmid using standard assembly methods<br />
Cut LacP-RBS1 using S <br />
Cut CHS3 using X and S<br />
(50% chance of inserting backwards --> must select for this after)<br />
----<br />
<br />
===9/10/10===<br />
Inoculated 20 LacP-RBS-CHS3 cells.<br />
<br />
Assembled CP-LacPI-RFP parts into Tet backbone.<br />
----</div>Gcxmatthttp://2010.igem.org/Week13_9/5/10-9/11/10Week13 9/5/10-9/11/102010-10-27T02:10:35Z<p>Gcxmatt: /* 9/7/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week13 Highlights===<br />
We began reorganizing our lab space and equipment in preparation for a fall quarter class that would be utilizing the majority of the lab. We attempted standard assembly of LacP-RBS-CHS3 because the presence of a P site in CHS3 prevented 3A assembly. <br />
----<br />
===9/6/10===<br />
Kevin did some reorganizing to get the lab back in order for Prof. Mordacq's class this fall. He consolidated the bench space, the -20 refrigerator and the cold room.<br />
<br />
===9/7/10===<br />
Day Off<br />
----<br />
<br />
===9/8/10===<br />
********<br />
----<br />
===9/9/10===<br />
Assembled LacP-RBS1-CHS3 in a Chlor backbone plasmid using standard assembly methods<br />
Cut LacP-RBS1 using S <br />
Cut CHS3 using X and S<br />
(50% chance of inserting backwards --> must select for this after)<br />
----<br />
<br />
===9/10/10===<br />
Inoculated 20 LacP-RBS-CHS3 cells.<br />
<br />
Assembled CP-LacPI-RFP parts into Tet backbone.<br />
----</div>Gcxmatthttp://2010.igem.org/Week12_8/29/10-9/4/10Week12 8/29/10-9/4/102010-10-27T02:10:08Z<p>Gcxmatt: /* 9/1/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week12 Highlights===<br />
We began assembling LacP-RBS-CHS3. We defrosted our -20 and in the process reorganized the -20 and threw out unnecessary DNA.<br />
----<br />
<br />
===8/30/10===<br />
<br />
Screened MAGE culture for knockouts.<br />
<br />
----<br />
<br />
===8/31/10===<br />
Attempted to assemble LacP-RBS-CHS3 --> accidentally cut CHS3 with P (contains a P site)<br />
<br />
Reorganized the racks in the -20. <br />
<br />
Threw out unnecessary/old DNA from our -20.<br />
<br />
Moved all of our racks from our -20 to another -20 in order to defrost our -20.<br />
<br />
tqsA Knockouts FOUND (?) in MAGE culture upon screening.<br />
----<br />
<br />
===9/1/10===<br />
Ragan left for home today and left MAGE in our hands. He asked us to test his tsqA knockouts to determine if they were in fact true knockouts.<br />
<br />
----<br />
<br />
===9/2/10===<br />
Day Off<br />
----<br />
<br />
===9/3/10===<br />
Reorganized -20 DNA stock. Created racks for individual teammates to stay organized once the school year starts.<br />
----</div>Gcxmatthttp://2010.igem.org/Week12_8/29/10-9/4/10Week12 8/29/10-9/4/102010-10-27T02:09:53Z<p>Gcxmatt: /* 9/2/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week12 Highlights===<br />
We began assembling LacP-RBS-CHS3. We defrosted our -20 and in the process reorganized the -20 and threw out unnecessary DNA.<br />
----<br />
<br />
===8/30/10===<br />
<br />
Screened MAGE culture for knockouts.<br />
<br />
----<br />
<br />
===8/31/10===<br />
Attempted to assemble LacP-RBS-CHS3 --> accidentally cut CHS3 with P (contains a P site)<br />
<br />
Reorganized the racks in the -20. <br />
<br />
Threw out unnecessary/old DNA from our -20.<br />
<br />
Moved all of our racks from our -20 to another -20 in order to defrost our -20.<br />
<br />
tqsA Knockouts FOUND (?) in MAGE culture upon screening.<br />
----<br />
<br />
===9/1/10===<br />
Ragan left for home today and left MAGE in our hands. He asked us to test his tsqA knockouts to determine if they were in fact true knockouts.<br />
<br />
===9/2/10===<br />
Day Off<br />
----<br />
<br />
===9/3/10===<br />
Reorganized -20 DNA stock. Created racks for individual teammates to stay organized once the school year starts.<br />
----</div>Gcxmatthttp://2010.igem.org/Week9_8/8/10-8/14/10Week9 8/8/10-8/14/102010-10-27T02:09:24Z<p>Gcxmatt: /* 8/13/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week9 Highlights===<br />
We bought chitosan powder form a local health store to use as a positive control for chitin staining. We ran a colony PCR on pMAL-CHS3 and found no positive results. We began MAGE knockouts of tqsA. Discovered that our Holin1, Holin2, pMAL-CHS3, and CP-LacPi parts were faulty. We also decided to use a rhodamine-conjugated chitin probe as an alternative chitin stain because calcofluor white resulted in excess background.<br />
<br />
----<br />
<br />
===8/9/10===<br />
Bought Chitosan powder to use as a positive control for calcofluor white --> glowed bright blue/green when stained<br />
<br />
Stained (pMal-CHS3) ligation products for the presence of chitin --> ligation 1/tube 3 had slight fluorescence <br />
<br />
Used UV-vis Spectroscopy to measure chitin concentrations <br />
<br />
Grew bakers yeast to test for presence of chitin<br />
<br />
Colony PCR on (pMal-CHS3)<br />
<br />
----<br />
<br />
===8/10/10===<br />
Ran gel for colony PCR no positives<br />
<br />
Ran PCR for CHS3 insert into pMal vector<br />
<br />
Stained varying concentrations of chitosan (.25g/ml, 25ug/ml, 5ug/ml, 1ug/ml) and (pMal-CHS3) cells --> cells showed no signs of chitin<br />
<br />
Ligated (CP-LacPi)-(GFP) in Tet backbone<br />
<br />
Made LB minimum media for electroporated cells<br />
<br />
Miniprepped circular backbone plasmid<br />
----<br />
<br />
===8/11/10===<br />
Digested and ran a gel of CHS3 and backbone plasmids (C,T,K,A)<br />
<br />
Tested restriction enzymes by digesting circular plasmids with each enzyme individually --> saw 1 2500kb band which was expected<br />
<br />
Organized the DNA samples in our -20.<br />
----<br />
===8/12/10===<br />
Weekly meeting<br />
*Went over our gels --> identified faulty parts (holin1, holin2, CHS3-pMal, CP-LacPI)<br />
*Plan to re-kit to stock and reassemble<br />
<br />
Decided to use an alternative stain because calcofluor results in too much background and our confocal microscope can not excite in the UV spectrum<br />
----<br />
===8/13/10===<br />
Methanol fixation of yeast and E.Coli cells --> stained with rhodamine-conjugated chitin probe (allowed to incubate overnight)<br />
<br />
Competency of ChiA knockouts = 2.5x10^7 and 5.0x10^6<br />
----</div>Gcxmatthttp://2010.igem.org/Week7_7/25/10-7/31/10Week7 7/25/10-7/31/102010-10-27T02:08:54Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week7 Highlights===<br />
Half of our team received BIF confocal microscopy training. We determined that our Amp plates were bad because of excess growth occurring on our overnight transformation plates. We began PCRing our pMAL vector. We continued having problems with 3A assembly. We tried using 1ug of starting DNA instead of 500ng. We also tried following Knight's 3A assembly protocol. All of our assemblies still failed.<br />
<br />
----<br />
<br />
===7/26/10===<br />
BIF confocal microscopy training<br />
<br />
Kit to stock of 1-6G and 1-12O<br />
<br />
Ran a gel of the pMAL PCR products --> very faint bands<br />
<br />
Used pMAL PCR products as template for another PCR reaction<br />
<br />
Ligated LacP1-GFP and transformed using our own TOP10 competent cells<br />
<br />
----<br />
<br />
===7/27/10===<br />
Excessive growth of 1-6G and 1-12O plates --> amp plates are possibly bad<br />
<br />
Gel extraction LacP1, GFP, Linear Tet backbone plasmid restriction digest products<br />
<br />
Ran a gel of digestion and ligation products; conclusion: linear plasmid PCR products are bad. Also threw out other assortment of degraded DNA.<br />
<br />
3A Assembly using 1 ug of starting DNA.<br />
<br />
Poured new Tet and Chl plates<br />
<br />
----<br />
===7/28/10===<br />
Followed Knight's 3A Assembly for LacP1-GFP part --> No growth<br />
----<br />
<br />
===7/29/10===<br />
3A Assembly using Knight protocol restriction digests --> but replaced linear backbone plasmid with circular plasmid miniprepped from IGEM kit<br />
<br />
----<br />
<br />
===7/30/10===<br />
LACP1 with GFP in CP-C yielded good results<br />
<br />
Ligations (CP1, LACP1, LACPI1 with GFP1 in CP-C)<br />
<br />
Redid PCR of CHS3 for pMAL insert<br />
----</div>Gcxmatthttp://2010.igem.org/Week4_7/4/10-7/10/10Week4 7/4/10-7/10/102010-10-27T02:08:34Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week4 Highlights===<br />
We changed our overnight culture protocol to 13 hours instead of 18 hours, but we still have problems with low DNA yields. We thought maybe our antibiotic levels were to blame. Our first PCR reaction of CHS3 failed. We met with Peggy Saks and learned about the Keio Collection where we can find ChiA and ydgG knockouts. We also purchased our IPTG spray bottles.<br />
<br />
----<br />
<br />
===7/4/10===<br />
13hr(2ml) -> miniprep -> ~45ng/μl<br />
<br />
13hr(3ml) -> miniprep -> ~55ng/μl<br />
<br />
15hr(2ml) -> miniprep -> 40ng/μl<br />
<br />
Also, NB consistently gave higher yields than T10 by 5~10ng/μl<br />
<br />
Note: 18hr gave higher yields than either 13hr or 15hr<br />
<br />
Protocol: 18hr, NB, 5ml incubation (spin all down)<br />
----<br />
<br />
===7/5/10===<br />
CHS3 Gel Extraction -> 8ng/μl<br />
<br />
Restarted CHS3 PCR added 5 cycles.<br />
<br />
Kit to Stock:<br />
<br />
*'''2-8E''' (j06702: RFP)<br />
*'''1-18C''' (j23100: CP1)<br />
*'''1-18K''' (j23104: CP2)<br />
*'''1-18M''' (j23105: CP3)<br />
*'''1-2M''' (b0034: RBS1)<br />
*'''1-2G''' (b0031: RBS2)<br />
*'''1-2I''' (b0032: RBS3)<br />
*'''1-23L'''(b0015: DT1)<br />
*'''1-2O''' (c0012: LacL)<br />
<br />
Reincubated (5μl transfer to liquid culture) 1-12O, 1-12M, 1-6G (GFP1, GFP2, LacP1 respectively)<br />
<br />
Notes: Need Cells; Reorganize Lab (assign benches)<br />
----<br />
<br />
===7/6/10===<br />
Miniprep (16hr/5ml) -> around 50ng/μl<br />
<br />
Ran another gel extraction for CHS3<br />
<br />
Kit to Stock:<br />
*'''1-1D''' (r0010: LacP2)<br />
*'''3-20K''' (K124014: Holin2)<br />
<br />
Plated 1-1D, 3-20K and incubated overnight.<br />
<br />
Colonies were selected from the NB-8E, NB-18C, NB-18K, NB-18M, NB-2M, NB-2G, T10-2I, T10-23L, T10-6K, and T10-2O plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 6:00pm).<br />
----<br />
<br />
===7/7/10===<br />
Miniprep (25ul of Amp/5ml spin down) --> 3 ~100ng/μl and 2 ~180ng/μl <br />
<br />
Miniprep (50ul of Amp/1.5ml spin down) --> all ~20ng/μl<br />
<br />
Miniprep (50ul of Amp/3.5ml spin down) --> all ~20ng/μl<br />
<br />
50μg/ml of Amp = seems to result in more DNA (half the concentration of what we previously used)<br />
<br />
Gel extraction --> very bright CHS3 band (10.1ng/μl)<br />
<br />
Colonies were selected from the T10-2I, T10-23L, T10-6K, T10-2O, T10-1D, T10-20K plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 7:30pm)<br />
<br />
----<br />
<br />
===7/8/10===<br />
Ethanol precipitation of 1-6G, 2-8E, 1-12O, and 1-12M<br />
<br />
Keio Collection: ChiA knockouts grew, but YdgG knockouts did not (possibly dead due to storage in -20 instead of -80)<br />
<br />
Started another PCR reaction of CHS3<br />
<br />
Miniprep (5ml spin down/25ul of antibiotic) --> mostly ~50ng/ul<br />
<br />
Miniprep (5ml spin down/20ul of antibiotic) --> mostly ~70ng/ul<br />
<br />
Diagrammed out our assembly methods for the CHS3 plasmid and the Apoptotic plasmid<br />
<br />
----<br />
<br />
===7/9/10===<br />
Finished the ethanol precipiation (RESULTS)<br />
<br />
Started assembling the LacP-RFP, LacP-GFP1, and LacP-GFP2 pieces for use in testing the Lac promoter.<br />
<br />
----<br />
===7/10/10===<br />
IPTG Spray Bottles:<br />
10 Sprays = ~1.25ml --> .125ml/spray<br />
100mM IPTG Stpcl = 1 gram/41.96ml<br />
<br />
3 Sprays of 1mM IPTG (started at 5:15pm)<br />
*30 Min<br />
*1 Hour<br />
<br />
3 Sprays of 5mM IPTG (started at 5:15pm)<br />
*30 Min<br />
*1 Hour<br />
----</div>Gcxmatthttp://2010.igem.org/Week2_6/20/10-6/26/10Week2 6/20/10-6/26/102010-10-27T02:08:16Z<p>Gcxmatt: /* 6/22/10 */</p>
<hr />
<div>__NOTOC__<br />
===Week2 Highlights===<br />
We began making antibiotic plates. We also took out parts 12O and 6G from the iGEM kit because we planned on testing inducible promoters. We ran into our first problem when we received low DNA yields after DNA extraction. We also experienced weird reddish/pink colonies in our 12O plates which we expected to be completely white.<br />
<br />
----<br />
<br />
===6/22/10===<br />
First day of real lab work.<br />
<br />
We poured our Kan, Amp, Tet, Kan+Amp, Kan+Tet, Amp+Tet antibiotic agar plates.<br />
<br />
<u>Antibiotic Concentrations</u><br />
*Amp: 50mg/500ml <br />
<br />
*Kan: 31.97mg/500ml<br />
<br />
*Tet: 6mg/500ml<br />
<br />
----<br />
<br />
===6/23/10===<br />
Resuspended 12O and 6G DNA from the iGEM Kit and transformed them into our competent cells.<br />
<br />
*'''6G''' = r0011(inducible promoter)<br />
*'''12O''' = e0840(rbs30-gfp-2xterm)<br />
<br />
----<br />
<br />
===6/24/10===<br />
Ran a gel of our PCR product to make sure that our taq polymerase master mix was working. <br />
<br />
Selected single 12O and 6G colonies and transferred them to LB and incubated for 18 hours.<br />
<br />
Let 1 12O and 1 6G plate incubate at RT. Let 1 12O and 1 6G plate incubate at 37°C. Intended to observe lawn growth.<br />
<br />
----<br />
<br />
===6/25/10===<br />
Low DNA yield; Plan to redo DNA extraction from Kit<br />
<br />
Found red/pink colonies in 12O plates. Thought to be contaminations. Plates were thrown out.<br />
<br />
----</div>Gcxmatthttp://2010.igem.org/Week2_6/20/10-6/26/10Week2 6/20/10-6/26/102010-10-27T02:07:46Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week2 Highlights===<br />
We began making antibiotic plates. We also took out parts 12O and 6G from the iGEM kit because we planned on testing inducible promoters. We ran into our first problem when we received low DNA yields after DNA extraction. We also experienced weird reddish/pink colonies in our 12O plates which we expected to be completely white.<br />
<br />
----<br />
<br />
===6/22/10===<br />
<br />
We poured our Kan, Amp, Tet, Kan+Amp, Kan+Tet, Amp+Tet antibiotic agar plates.<br />
<br />
<u>Antibiotic Concentrations</u><br />
*Amp: 50mg/500ml <br />
<br />
*Kan: 31.97mg/500ml<br />
<br />
*Tet: 6mg/500ml<br />
<br />
----<br />
<br />
===6/23/10===<br />
Resuspended 12O and 6G DNA from the iGEM Kit and transformed them into our competent cells.<br />
<br />
*'''6G''' = r0011(inducible promoter)<br />
*'''12O''' = e0840(rbs30-gfp-2xterm)<br />
<br />
----<br />
<br />
===6/24/10===<br />
Ran a gel of our PCR product to make sure that our taq polymerase master mix was working. <br />
<br />
Selected single 12O and 6G colonies and transferred them to LB and incubated for 18 hours.<br />
<br />
Let 1 12O and 1 6G plate incubate at RT. Let 1 12O and 1 6G plate incubate at 37°C. Intended to observe lawn growth.<br />
<br />
----<br />
<br />
===6/25/10===<br />
Low DNA yield; Plan to redo DNA extraction from Kit<br />
<br />
Found red/pink colonies in 12O plates. Thought to be contaminations. Plates were thrown out.<br />
<br />
----</div>Gcxmatthttp://2010.igem.org/Week20_10/24/10-10/30/10Week20 10/24/10-10/30/102010-10-27T02:05:36Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week20 Highlights===<br />
We continued our efforts to assembly CP-LacPI-GFP in hopes that we can use the plate readers to characterize the parts before the Jamboree. This was the week of the WIKI FREEZE.<br />
----<br />
<br />
===10/24/10===<br />
Timi took out the CP-LacPI plates into the morning and stored them in the cold room. <br />
<br />
Kevin inoculated overnight cultures and put them in to grow.<br />
----<br />
<br />
===10/25/10===<br />
Kevin miniprepped the CP-LacPI cultures and digested them.<br />
----<br />
<br />
===10/26/10===<br />
Timi treated Tet backbone with phosphatase for more efficient 3A assemblies. <br />
<br />
Timi ligated the CP-LacPI digests with GFP into a Tet backbone. They were transformed into ChiA competent cells and plated onto Tet plates.<br />
----<br />
<br />
===10/27/10===<br />
Kevin inoculated overnight cultures of the CP-LacPI-GFP colonies. They will be miniprepped/digested tomorrow to be screen on an agarose gel. After we successfully obtain the part, we will characterize the CP-LacPI-GFP constructs that we created to find the weakest and strongest constitutive promoter. We will be using a plate reader to characterize the parts.<br />
<br />
WIKI FREEZE at 11:59PM ET. Any further work will be documented elsewhere.<br />
----</div>Gcxmatthttp://2010.igem.org/Week19_10/17/10-10/23/10Week19 10/17/10-10/23/102010-10-27T02:04:01Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week19 Highlights===<br />
We retried assembly of CP-LacPI both phosphatase treated and non-phosphatase treated. We were unable to assemble a full CP-LacPi-GFP part. We submitted 3 of our DNA constructs to the parts registry.<br />
----<br />
<br />
===10/17/10===<br />
Timi phosphotase-treated the newly digested chlor backbone and ligated a second set of CP-LacPI.<br />
Kevin transformed and plated this new set of phosophotase treated Cp-LacPI.<br />
<br />
Kevin inoculated the first set of non-phosphotase treated CP-LacPI inoculations.<br />
<br />
----<br />
<br />
===10/18/10===<br />
Matt miniprepped 31GFP, 21GFP (old), and 32GFP (old). He digested the miniprepped DNA with E/P for Kevin to run on a gel. He also reinoculated all 9 CP-LacPI combinations.<br />
<br />
Matt inoculated the phosophotase-treated ligations at 9pm.<br />
----<br />
<br />
===10/19/10===<br />
Plate reader with Kevin @ 3pm.<br />
<br />
Sean - ran CHS3(pMAL+SDM) digest, pMAL digest, and ligation on a gel -> CHS3 exhibited incorrect bands (there are 2, when there should be 1) ligation showed nothing -> plan to run CHS3 undigested and rerun the above 3.<br />
----<br />
<br />
===10/20/10===<br />
Timi and Kevin ligated the old 212, 312, and 322 parts into a phosophotase-treated Chlor backbone for submission into the registry. They transformed and plated for inoculation tomorrow night.<br />
<br />
They inoculated cultures for the plate reader.<br />
----<br />
<br />
===10/21/10===<br />
Timi/Kevin: Early morning plate reader protocol entering.<br />
<br />
Weekly lab meeting with advisors.<br />
----<br />
<br />
===10/22/10===<br />
PCR'ed out CHS3 with pMAL ends and Gel extracted<br />
<br />
Kevin was in lab until 3am to see if any of the CP-LacPI parts were ligated to GFP. He ran multiple gels and sent the results to Prof. Leonard. Unfortunately, we do not have the construct ready. Thus, we will likely not be able to characterize a CP-LacPI part by Wednesday. However, we plan on working to characterize one by the Jamboree.<br />
----<br />
<br />
===10/23/10===<br />
Timi miniprepped the overnights for DNA that will be submitted the registry. We will be sending in parts [[http://partsregistry.org/Part:BBa_K418003 BBa_K418003]], [[http://partsregistry.org/Part:BBa_K418005 BBa_K418005]] and [[http://partsregistry.org/Part:BBa_K418006 BBa_K418006]] on Monday afternoon via Fedex. <br />
<br />
Timi and Kevin retransformed the confirmed CP-LacPI parts (CP2-LacPI1, CP3-LacPI1 and CP3-LacPI2) from earlier in the summer into ChiA competent cells. The cells were plated onto Chlor plates. We will be continuing our work to hook them up to GFP over the subsequent weeks. Hopefully, we will have all of our work done by the Jamboree.<br />
----</div>Gcxmatthttp://2010.igem.org/Week18_10/10/10-10/16/10Week18 10/10/10-10/16/102010-10-27T01:59:26Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week18 Highlights===<br />
We discovered that there are 2 additional XbaI sites in our CHS3 gene while trying to enter our part into the iGEM website. We did not realize this before because we used NEB Cutter to analyze the gene and it did not indicate that and XbaI sites were present. XbaI sites only show up when you set the search parameters to specifically look for XbaI sites. This is very disappointing as digesting with X is an integral part of some of our planned assemblies.<br />
<br />
----<br />
<br />
===10/10/10===<br />
Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.<br />
----<br />
<br />
===10/11/10===<br />
Day Off<br />
----<br />
===10/12/10===<br />
Matt - digested LacP-RBS with SpeI in preparation for standard assembly with ES digested CHS3<br />
----<br />
<br />
===10/13/10===<br />
Day Off<br />
----<br />
===10/14/10===<br />
Kevin transformed T10 cells with CP-LacPI parts that we did not have much digest of left. He plated them onto TET plates.<br />
----<br />
<br />
===10/15/10===<br />
Kevin took out the CP-LacPI plates and put them into the 4 degree for Timi. Timi inoculated overnight cultures at 7pm.<br />
----<br />
<br />
===10/16/10===<br />
Team meeting at 12pm to work on our wiki. We made the discouraging discovery that our CHS3 DNA that Ben had worked super hard for actually contained XbaI restriction sites. This complicates our assembly and might jeopardize the success of our project. We will talk to the advisors about this to see if things can still work out.<br />
<br />
Timi and Kevin miniprepped the CP-LacPI inoculations, digested them with E&P, and ligated them to GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week18_10/10/10-10/16/10Week18 10/10/10-10/16/102010-10-27T01:59:09Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week18 Highlights===<br />
We discovered that there are 2 additional XbaI sites in our CHS3 gene while trying to enter our part into the iGEM website. We did not realize this before because we used NEB Cutter to analyze the gene and it did not indicate that and XbaI sites were present. XbaI sites only show up when you set the search parameters to specifically look for XbaI sites. This is very disappointing as digesting with X is an integral part of some of our planned assemblies.<br />
<br />
===10/10/10===<br />
Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.<br />
----<br />
<br />
===10/11/10===<br />
Day Off<br />
----<br />
===10/12/10===<br />
Matt - digested LacP-RBS with SpeI in preparation for standard assembly with ES digested CHS3<br />
----<br />
<br />
===10/13/10===<br />
Day Off<br />
----<br />
===10/14/10===<br />
Kevin transformed T10 cells with CP-LacPI parts that we did not have much digest of left. He plated them onto TET plates.<br />
----<br />
<br />
===10/15/10===<br />
Kevin took out the CP-LacPI plates and put them into the 4 degree for Timi. Timi inoculated overnight cultures at 7pm.<br />
----<br />
<br />
===10/16/10===<br />
Team meeting at 12pm to work on our wiki. We made the discouraging discovery that our CHS3 DNA that Ben had worked super hard for actually contained XbaI restriction sites. This complicates our assembly and might jeopardize the success of our project. We will talk to the advisors about this to see if things can still work out.<br />
<br />
Timi and Kevin miniprepped the CP-LacPI inoculations, digested them with E&P, and ligated them to GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week17_10/3/10-10/9/10Week17 10/3/10-10/9/102010-10-27T01:58:26Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week17 Highlights===<br />
We ran into problems while running the gels for our CP-LacPI-GFP parts. We decided to use new buffer.<br />
<br />
----<br />
===10/3/10===<br />
Timi miniprepped the CP-LacPI-GFP cultures and digested them with E/P. She then poured a big and a small gel.<br />
<br />
Kevin came in and ran the digested CP-LacPI-GFP, digested CP-LacPI with digested Cp and digested LacPI parts.<br />
----<br />
<br />
===10/4/10===<br />
Day Off<br />
----<br />
<br />
===10/5/10===<br />
Timi and Kevin came in to discuss why their gels were running strangely. We believe there is something wrong with our buffer, so we are swapping everything out with a fresh batch of Prof. Mordacq's 0.5x TBE buffer. Timi poured a large gel that we will be using tomorrow.<br />
----<br />
===10/6/10===<br />
Day Off<br />
----<br />
===10/7/10===<br />
Timi- took part 2-17F out of the DNA kit. Transformed into T10 cells to grow overnight.<br />
----<br />
<br />
===10/8/10===<br />
Kevin- ran gel of the CP-LacPI parts. Gel results are still inconclusive. Also put in an overnight culture of the 2-17F part into the incubator for Timi.<br />
----<br />
<br />
===10/9/10===<br />
Timi- Miniprepped the 2-17F part. Digested the DNA with E/P and ligated the 2-17F GFP part into Chlor and Tet backbone.<br />
----</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T01:56:05Z<p>Gcxmatt: /* Daily Accomplishments */</p>
<hr />
<div>__NOTOC__<br />
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
=='''Notebook'''==<br />
<br />
=='''Weekly Accomplishments'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-27T01:55:55Z<p>Gcxmatt: </p>
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]<br />
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]<br />
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]<br />
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]<br />
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]<br />
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]<br />
!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]<br />
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]<br />
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
=='''Notebook'''==<br />
<br />
=='''Weekly Accomplishments'''==<br />
[[Brainstorming April-June 2010]]<br />
<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
=='''Daily Accomplishments'''==<br />
<br />
<br />
<br />
<br />
<br />
<br />
|}</div>Gcxmatthttp://2010.igem.org/Week20_10/24/10-10/30/10Week20 10/24/10-10/30/102010-10-27T01:55:52Z<p>Gcxmatt: New page: ===10/24/10=== Timi took out the CP-LacPI plates into the morning and stored them in the cold room. Kevin inoculated overnight cultures and put them in to grow. ---- ===10/25/10=== Kevi...</p>
<hr />
<div>===10/24/10===<br />
Timi took out the CP-LacPI plates into the morning and stored them in the cold room. <br />
<br />
Kevin inoculated overnight cultures and put them in to grow.<br />
----<br />
<br />
===10/25/10===<br />
Kevin miniprepped the CP-LacPI cultures and digested them.<br />
----<br />
<br />
===10/26/10===<br />
Timi treated Tet backbone with phosphatase for more efficient 3A assemblies. <br />
<br />
Timi ligated the CP-LacPI digests with GFP into a Tet backbone. They were transformed into ChiA competent cells and plated onto Tet plates.<br />
----<br />
<br />
===10/27/10===<br />
Kevin inoculated overnight cultures of the CP-LacPI-GFP colonies. They will be miniprepped/digested tomorrow to be screen on an agarose gel. After we successfully obtain the part, we will characterize the CP-LacPI-GFP constructs that we created to find the weakest and strongest constitutive promoter. We will be using a plate reader to characterize the parts.<br />
<br />
WIKI FREEZE at 11:59PM ET. Any further work will be documented elsewhere.<br />
----</div>Gcxmatthttp://2010.igem.org/Week19_10/17/10-10/23/10Week19 10/17/10-10/23/102010-10-27T01:55:18Z<p>Gcxmatt: New page: ===10/17/10=== Timi phosphotase-treated the newly digested chlor backbone and ligated a second set of CP-LacPI. Kevin transformed and plated this new set of phosophotase treated Cp-LacPI. ...</p>
<hr />
<div>===10/17/10===<br />
Timi phosphotase-treated the newly digested chlor backbone and ligated a second set of CP-LacPI.<br />
Kevin transformed and plated this new set of phosophotase treated Cp-LacPI.<br />
<br />
Kevin inoculated the first set of non-phosphotase treated CP-LacPI inoculations.<br />
<br />
----<br />
<br />
===10/18/10===<br />
Matt miniprepped 31GFP, 21GFP (old), and 32GFP (old). He digested the miniprepped DNA with E/P for Kevin to run on a gel. He also reinoculated all 9 CP-LacPI combinations.<br />
<br />
Matt inoculated the phosophotase-treated ligations at 9pm.<br />
----<br />
<br />
===10/19/10===<br />
Plan: Timi miniprepped the phosphotase-treated CP-LacPI and ran them on a gel. But Mordacq's class was using the thermocycler so Timi just sat and read.<br />
<br />
Plate reader fun with Kevin @ 3pm.<br />
<br />
Sean - ran CHS3(pMAL+SDM) digest, pMAL digest, and ligation on a gel -> CHS3 exhibited incorrect bands (there are 2, when there should be 1) ligation showed nothing -> plan to run CHS3 undigested and rerun the above 3.<br />
----<br />
<br />
===10/20/10===<br />
Timi and Kevin ligated the old 212, 312, and 322 parts into a phosophotase-treated Chlor backbone for submission into the registry. They transformed and plated for inoculation tomorrow night.<br />
<br />
They inoculated cultures for the plate reader.<br />
----<br />
<br />
===10/21/10===<br />
Timi/Kevin: Early morning plate reader protocol entering.<br />
<br />
Weekly lab meeting with advisors.<br />
----<br />
<br />
===10/22/10===<br />
PCR'ed out CHS3 with pMAL ends and Gel extracted<br />
<br />
Kevin was in lab until 3am to see if any of the CP-LacPI parts were ligated to GFP. He ran multiple gels and sent the results to Prof. Leonard. Unfortunately, we do not the have the construct ready. Thus, we will likely not be able to characterize a CP-LacPI part by Wednesday. However, we plan on working to characterize one by the Jamboree.<br />
----<br />
<br />
===10/23/10===<br />
Timi miniprepped the overnights for DNA that will be submitted the registry! We will be sending in parts [[http://partsregistry.org/Part:BBa_K418003 BBa_K418003]], [[http://partsregistry.org/Part:BBa_K418005 BBa_K418005]] and [[http://partsregistry.org/Part:BBa_K418006 BBa_K418006]] on Monday afternoon via Fedex. Yay!!<br />
<br />
Timi and Kevin retransformed the confirmed CP-LacPI parts (CP2-LacPI1, CP3-LacPI1 and CP3-LacPI2) from earlier in the summer into ChiA competent cells. The cells were plated onto Chlor plates. We will be continuing our work to hook them up to GFP over the subsequent weeks. Hopefully, we will have all of our work done by the Jamboree! Wish us luck!<br />
----</div>Gcxmatthttp://2010.igem.org/Week18_10/10/10-10/16/10Week18 10/10/10-10/16/102010-10-27T01:54:47Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week18 Highlights===<br />
We discovered that there are 2 additional XbaI sites in our CHS3 gene while trying to enter our part into the iGEM website. We did not realize this before because we used NEB Cutter to analyze the gene and it did not indicate that and XbaI sites were present. XbaI sites only show up when you set the search parameters to specifically look for XbaI sites. This is very disappointing as digesting with X is an integral part of some of our planned assemblies.<br />
<br />
===10/10/10===<br />
Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.<br />
----<br />
<br />
===10/11/10===<br />
----<br />
===10/12/10===<br />
Matt - digested LacP-RBS with SpeI in preparation for standard assembly with ES digested CHS3<br />
----<br />
<br />
===10/13/10===<br />
----<br />
===10/14/10===<br />
Kevin transformed T10 cells with CP-LacPI parts that we did not have much digest of left. He plated them onto TET plates.<br />
----<br />
<br />
===10/15/10===<br />
Kevin took out the CP-LacPI plates and put them into the 4 degree for Timi. Timi inoculated overnight cultures at 7pm.<br />
----<br />
<br />
===10/16/10===<br />
Team meeting at 12pm to work on our wiki. We made the discouraging discovery that our CHS3 DNA that Ben had worked super hard for actually contained XbaI restriction sites. This complicates our assembly and might jeopardize the success of our project. We will talk to the advisors about this to see if things can still work out.<br />
<br />
Timi and Kevin miniprepped the CP-LacPI inoculations, digested them with E&P, and ligated them to GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week18_10/10/10-10/16/10Week18 10/10/10-10/16/102010-10-27T01:54:32Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week18 Summary===<br />
We discovered that there are 2 additional XbaI sites in our CHS3 gene while trying to enter our part into the iGEM website. We did not realize this before because we used NEB Cutter to analyze the gene and it did not indicate that and XbaI sites were present. XbaI sites only show up when you set the search parameters to specifically look for XbaI sites. This is very disappointing as digesting with X is an integral part of some of our planned assemblies.<br />
<br />
===10/10/10===<br />
Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.<br />
----<br />
<br />
===10/11/10===<br />
----<br />
===10/12/10===<br />
Matt - digested LacP-RBS with SpeI in preparation for standard assembly with ES digested CHS3<br />
----<br />
<br />
===10/13/10===<br />
----<br />
===10/14/10===<br />
Kevin transformed T10 cells with CP-LacPI parts that we did not have much digest of left. He plated them onto TET plates.<br />
----<br />
<br />
===10/15/10===<br />
Kevin took out the CP-LacPI plates and put them into the 4 degree for Timi. Timi inoculated overnight cultures at 7pm.<br />
----<br />
<br />
===10/16/10===<br />
Team meeting at 12pm to work on our wiki. We made the discouraging discovery that our CHS3 DNA that Ben had worked super hard for actually contained XbaI restriction sites. This complicates our assembly and might jeopardize the success of our project. We will talk to the advisors about this to see if things can still work out.<br />
<br />
Timi and Kevin miniprepped the CP-LacPI inoculations, digested them with E&P, and ligated them to GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week17_10/3/10-10/9/10Week17 10/3/10-10/9/102010-10-27T01:53:39Z<p>Gcxmatt: New page: ===10/3/10=== Timi miniprepped the CP-LacPI-GFP cultures and digested them with E/P. She then poured a big and a small gel. Kevin came in and ran the digested CP-LacPI-GFP, digested CP-La...</p>
<hr />
<div>===10/3/10===<br />
Timi miniprepped the CP-LacPI-GFP cultures and digested them with E/P. She then poured a big and a small gel.<br />
<br />
Kevin came in and ran the digested CP-LacPI-GFP, digested CP-LacPI with digested Cp and digested LacPI parts.<br />
----<br />
<br />
===10/4/10===<br />
Timi and Kevin decided to take a day off :)<br />
----<br />
<br />
===10/5/10===<br />
Timi and Kevin came in to discuss why their gels were running strangely. We believe there is something wrong with our buffer, so we are swapping everything out with a fresh batch of Prof. Mordacq's 0.5x TBE buffer. Timi poured a large gel that we will be using tomorrow.<br />
----<br />
===10/6/10===<br />
----<br />
===10/7/10===<br />
Timi- took part 2-17F out of the DNA kit. Transformed into T10 cells to grow overnight.<br />
----<br />
<br />
===10/8/10===<br />
Kevin- ran gel of the CP-LacPI parts. Gel results are still inconclusive. Also put in an overnight culture of the 2-17F part into the incubator for Timi.<br />
----<br />
<br />
===10/9/10===<br />
Timi- Miniprepped the 2-17F part. Digested the DNA with E/P and ligated the 2-17F GFP part into Chlor and Tet backbone.<br />
----</div>Gcxmatthttp://2010.igem.org/Team:Northwestern/NotebookTeam:Northwestern/Notebook2010-10-19T00:12:59Z<p>Gcxmatt: /* 10/16/10 */</p>
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<br />
[[Image:LOGOBANNER1.jpg|1000px|center|link=http://wikipedia.org|Tech Institute]]<br />
<br />
<!--- The Mission, Experiments ---><br />
{|- style="background: "transparent"; style="font-size: 87%;" color:#000000" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"<br />
!align="center"|[[Team:Northwestern|<span style="color:#000000;">'''Home'''</span>]]<br />
!align="center"|[[Team:Northwestern/Brainstorm|<span style="color:#000000;">'''Brainstorm'''</span>]]<br />
!align="center"|[[Team:Northwestern/Team|<span style="color:#000000;">'''Team'''</span>]]<br />
!align="center"|[[Team:Northwestern/Acknowledgements|<span style="color:#000000;">'''Acknowledgements'''</span>]]<br />
!align="center"|[[Team:Northwestern/Project|<span style="color:#000000;">'''Project'''</span>]]<br />
!align="center"|[[Team:Northwestern/SideProject|<span style="color:#000000;">'''Human Practices'''</span>]]<br />
!align="center"|[[Team:Northwestern/Parts|<span style="color:#000000;">'''Parts'''</span>]]<br />
!align="center"|[[Team:Northwestern/Notebook|<span style="color:#000000;">'''Notebook'''</span>]]<br />
!align="center"|[[Team:Northwestern/Calendar|<span style="color:#000000;">'''Calendar'''</span>]]<br />
!align="center"|[[Team:Northwestern/Protocol|<span style="color:#000000;">'''Protocol'''</span>]]<br />
!align="center"|[[Team:Northwestern/Safety|<span style="color:#000000;">'''Safety'''</span>]]<br />
!align="center"|[[Team:Northwestern/Links|<span style="color:#000000;">'''Links'''</span>]]<br />
!align="center"|[[Team:Northwestern/References|<span style="color:#000000;">'''References'''</span>]]<br />
!align="center"|[[Team:Northwestern/Media|<span style="color:#000000;">'''Media'''</span>]]<br />
!align="center"|[[Team:Northwestern/Contact|<span style="color:#000000;">'''Contact'''</span>]]<br />
|}<br />
<br />
{| align="center" border="0" width="75%"<br />
|<br />
<br />
[[Brainstorming April-June 2010]]<br />
<br />
[[Week1 6/13/10-6/19/10]]<br />
<br />
[[Week2 6/20/10-6/26/10]]<br />
<br />
[[Week3 6/27/10-7/3/10]]<br />
<br />
[[Week4 7/4/10-7/10/10]]<br />
<br />
[[Week5 7/11/10-7/17/10]]<br />
<br />
[[Week6 7/18/10-7/24/10]]<br />
<br />
[[Week7 7/25/10-7/31/10]]<br />
<br />
[[Week8 8/1/10-8/7/10]]<br />
<br />
[[Week9 8/8/10-8/14/10]]<br />
<br />
[[Week10 8/15/10-8/21/10]]<br />
<br />
[[Week11 8/22/10-8/28/10]]<br />
<br />
[[Week12 8/29/10-9/4/10]]<br />
<br />
[[Week13 9/5/10-9/11/10]]<br />
<br />
[[Week14 9/12/10-9/18/10]]<br />
<br />
[[Week15 9/19/10-9/25/10]]<br />
<br />
[[Week16 9/26/10-10/2/10]]<br />
<br />
[[Week17 10/3/10-10/9/10]]<br />
<br />
[[Week18 10/10/10-10/16/10]]<br />
<br />
[[Week19 10/17/10-10/23/10]]<br />
<br />
[[Week20 10/24/10-10/30/10]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
===10/3/10===<br />
Timi miniprepped the CP-LacPI-GFP cultures and digested them with E/P. She then poured a big and a small gel.<br />
<br />
Kevin came in and ran the digested CP-LacPI-GFP, digested CP-LacPI with digested Cp and digested LacPI parts.<br />
----<br />
<br />
===10/4/10===<br />
Timi and Kevin decided to take a day off :)<br />
----<br />
<br />
===10/5/10===<br />
Timi and Kevin came in to discuss why their gels were running strangely. We believe there is something wrong with our buffer, so we are swapping everything out with a fresh batch of Prof. Mordacq's 0.5x TBE buffer. Timi poured a large gel that we will be using tomorrow.<br />
----<br />
===10/6/10===<br />
----<br />
===10/7/10===<br />
Timi- took part 2-17F out of the DNA kit. Transformed into T10 cells to grow overnight.<br />
----<br />
<br />
===10/8/10===<br />
Kevin- ran gel of the CP-LacPI parts. Gel results are still inconclusive. Also put in an overnight culture of the 2-17F part into the incubator for Timi.<br />
----<br />
<br />
===10/9/10===<br />
Timi- Miniprepped the 2-17F part. Digested the DNA with E/P and ligated the 2-17F GFP part into Chlor and Tet backbone.<br />
----<br />
<br />
===10/10/10===<br />
Timi- Transformed and plated part 2-17F onto Chlor and Tet plates. Will be ready for miniprepping tomorrow.<br />
----<br />
<br />
===10/11/10===<br />
----<br />
===10/12/10===<br />
Matt - digested LacP-RBS with SpeI in preparation for standard assembly with ES digested CHS3<br />
----<br />
<br />
===10/13/10===<br />
----<br />
===10/14/10===<br />
Kevin transformed T10 cells with CP-LacPI parts that we did not have much digest of left. He plated them onto TET plates.<br />
----<br />
<br />
===10/15/10===<br />
Kevin took out the CP-LacPI plates and put them into the 4 degree for Timi. Timi inoculated overnight cultures at 7pm.<br />
----<br />
<br />
===10/16/10===<br />
Timi and Kevin miniprepped the CP-LacPI inoculations, digested them with ___, and ligated them to GFP.<br />
----<br />
===10/18/10===<br />
Matt miniprepped 31GFP, 21GFP (old), and 32GFP (old). He digested the miniprepped DNA with E/P for Kevin to run on a gel. He also reinoculated all 9 CP-LacPI combinations.<br />
----<br />
|}</div>Gcxmatthttp://2010.igem.org/Week16_9/26/10-10/2/10Week16 9/26/10-10/2/102010-10-16T23:18:27Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week16 Highlights===<br />
We continued work with Cp-LacPi-GFP constructs, but failed to get Cp-LacPi parts based on our gels. We also realized that our pMAL primers were incorrect so we placed an order for new primers. We completed site directed mutagenesis of CHS3.<br />
<br />
----<br />
===9/26/10===<br />
Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!<br />
<br />
Ran gels for the digestions --> no band was less than 1000 bp (Cp-LacPi1 parts should be ~90bp and Cp-LacPi2 parts should be ~235bp)<br />
<br />
Is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid? <br />
<br />
'''The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look.''' (REMOVE THIS LATER)<br />
<br />
The bands in the gel were also a little wavy. According to Professor Mordacq, this can happen when the agarose is not properly melted all the way. <br />
<br />
Innoculated the CP-LacPi-GFP parts again.<br />
<br />
===9/27/10===<br />
Inserted LacP-RBS into Tet plasmid --> transformed into ChiA knockouts<br />
<br />
Kevin went in between classes to spin down the incubations from last night.<br />
<br />
Timi picked up from there and miniprepped the samples and then digested them.<br />
----<br />
<br />
===9/28/10===<br />
pMAL - realized primers are bad --> reordering correct ones<br />
<br />
Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.<br />
<br />
Tried Cp-LacPi digestions using more DNA --> still too difficult to visualize the small inserts<br />
<br />
*This could be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.<br />
<br />
----<br />
<br />
===9/29/10===<br />
Site-directed mutagenesis: Amplification and DpnI digestion completed.<br />
<br />
Ragan had some trouble with the TBE buffer. We are looking into what went wrong with it. Sean?<br />
----<br />
<br />
===9/30/10===<br />
Transformation for Site-Directed mutagenesis completed.<br />
<br />
At the lab meeting this morning, Timi discovered that Kevin had been looking for an incorrectly-sized band in the gels. They went back through the photos of gels later in the day, but were inconclusive. They are going to back-track a little bit to figure out if any of the colonies that they had picked were actually positive.<br />
----<br />
<br />
===10/1/10===<br />
The transformation resulted in a good number of colonies for site-directed mutagenesis. 10 liquid cultures were made.<br />
<br />
Timi went in early to pour a big and small gel. BUT she forgot to put in the combs. FAIL.<br />
<br />
Kevin repoured the gels and ran the digested DNA on it. However, he forgot to run the digested parts on it as well, as the advisors had suggested.<br />
----<br />
<br />
===10/2/10===<br />
Timi and Kevin put in 15 overnight inculations of 31-GFP, 32-GFP and 21-GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week15_9/19/10-9/25/10Week15 9/19/10-9/25/102010-10-16T23:07:32Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week15 Highlights===<br />
Classes at Northwestern started this week. This complicated our functionality as a team due to scheduling. Thus, we paired up and began working on different portions of the project on our own.<br />
<br />
*Timi/Kevin: CP-LacPI assembly<br />
<br />
*Matt/Sean: Chitin synthase<br />
<br />
*Ben: Site directed mutagenesis<br />
<br />
*Ragan: MAGE<br />
<br />
We submitted our iGEM abstract and has a group meeting for the first time since everyone left to go back home for a few weeks. We realized that something was wrong with our originally PCRed CHS3, so we remade it. However, we were only able to get 25-35 ng/ul of DNA. We also began an extra weekly meeting on Saturdays around 4pm to work on our poster, presentation, and wiki pages.<br />
<br />
----<br />
<br />
===9/20/10===<br />
iGEM abstracts were due tonight. We had an after-hours lab meeting to finish things up. Prof. Leonard stopped by to help us make some final edits and to figure out how much money we had left in the budget.<br />
<br />
We also took this time to catch eachother up on what we had been working on since team members started leaving for home.<br />
----<br />
<br />
===9/21/10===<br />
Timi and Kevin poured a large gel prior to heading home for the night.<br />
----<br />
<br />
===9/22/10===<br />
Timi and Kevin loaded digested CP-LacPI assemblies into gels this morning and Kevin ran the gel between classes.<br />
----<br />
<br />
===9/23/10===<br />
Early 8am team meeting with Prof. Mordacq.<br />
<br />
CHS3 made, low conc (25-35) 5 tubes of about 80ul each in 4degree<br />
----<br />
<br />
===9/24/10===<br />
Digested LacP-RBS(2) with ES.<br />
Digested Tet plasmid with ES.<br />
----<br />
===9/25/10===<br />
We had a group lab meeting to begin working on the poster and presentation.<br />
<br />
Timi and Kevin put 18 overnight cultures of CP-LacPI assemblies into the incubator with Tet antibiotic (UHOH!).<br />
<br />
The Wildcats won their game! 4-0! Go cats! :)<br />
----</div>Gcxmatthttp://2010.igem.org/Week14_9/12/10-9/18/10Week14 9/12/10-9/18/102010-10-16T23:07:16Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week14 Highlights===<br />
We ran multiple gels to test our LacP-RBS and LacP-RBS-CHS3 assemblies. We continued preparing and assembling our combinations of Cp-LacPi-GFP for characterization. We also poured more Chlor and Tet plates using smaller (60mm x 15mm) petri dishes given to us by VWR.<br />
----<br />
===9/13/10===<br />
Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.<br />
----<br />
<br />
===9/14/10===<br />
Inoculated Apop1<br />
<br />
Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes<br />
<br />
Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.<br />
----<br />
<br />
===9/15/10===<br />
Miniprepped LacP-RBS-CHS3 and Apop1<br />
<br />
Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)<br />
<br />
Kevin put 15 overnight cultures into the 37 degree incubator at 6pm. <br />
<br />
Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate). <br />
<br />
Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.<br />
----<br />
<br />
===9/16/10===<br />
Ran digested LacP-RBS-CHS3 on a gel:<br />
*Majority contained 1 band around 3600bp<br />
*3 contained 3 bands (2000bp, 1600bp, 1100bp)<br />
<br />
Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.<br />
----<br />
<br />
===9/17/10===<br />
Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel<br />
LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp<br />
<br />
Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.<br />
<br />
Anyhow- results! Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.<br />
<br />
CP3-LacPi2 retrans:<br />
<br />
1) 155.3<br />
<br />
2) 135.1<br />
<br />
3) 76.8<br />
<br />
CP1-LacPi2 seq:<br />
<br />
1) 102.9<br />
<br />
2) 91.7<br />
<br />
3) 71.7<br />
<br />
CP2-LacPi2:<br />
<br />
1) 83.8<br />
<br />
2) 89.6<br />
<br />
CP1-LacPi2<br />
<br />
1) 54.7<br />
<br />
2) 94.7<br />
<br />
CP1-LacPi1:<br />
<br />
1) 108.8<br />
<br />
2) 66.0<br />
<br />
3) 36.5<br />
<br />
The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.<br />
<br />
<br />
PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.<br />
<br />
Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.<br />
----</div>Gcxmatthttp://2010.igem.org/Week13_9/5/10-9/11/10Week13 9/5/10-9/11/102010-10-16T23:07:10Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week13 Highlights===<br />
We began reorganizing our lab space and equipment in preparation for a fall quarter class that would be utilizing the majority of the lab. We attempted standard assembly of LacP-RBS-CHS3 because the presence of a P site in CHS3 prevented 3A assembly. <br />
----<br />
===9/6/10===<br />
Kevin did some reorganizing to get the lab back in order for Prof. Mordacq's class this fall. He consolidated the bench space, the -20 refrigerator and the cold room.<br />
<br />
===9/7/10===<br />
********<br />
----<br />
===9/8/10===<br />
********<br />
----<br />
===9/9/10===<br />
Assembled LacP-RBS1-CHS3 in a Chlor backbone plasmid using standard assembly methods<br />
Cut LacP-RBS1 using S <br />
Cut CHS3 using X and S<br />
(50% chance of inserting backwards --> must select for this after)<br />
----<br />
<br />
===9/10/10===<br />
Inoculated 20 LacP-RBS-CHS3 cells.<br />
<br />
Assembled CP-LacPI-RFP parts into Tet backbone.<br />
----</div>Gcxmatthttp://2010.igem.org/Week10_8/15/10-8/21/10Week10 8/15/10-8/21/102010-10-16T23:06:55Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week10 Highlights===<br />
We continued working on the MAGE tqsA knockouts. We cloned CHS3 into a standard chlor biobricks backbone and began site directed mutagenesis (SDM) to remove the PstI site.<br />
<br />
----<br />
<br />
===8/16/10===<br />
Cloned CHS3 into Chlor Biobricks plasmid --> will be used for Site Directed Mutagenesis<br />
<br />
Cycles 2 and 3 of MAGE for tqsA Knockouts.<br />
<br />
----<br />
<br />
===8/17/10===<br />
Site-Directed Mutagenesis to remove the PstI site in CHS3<br />
<br />
Cycles 4-6 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/18/10===<br />
Site-Directed Mutagenesis. Ran screening for 8/17, none were positive so repeated reaction with better protocol. (PCR only)<br />
<br />
Cycle 7 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/19/10===<br />
Site-Directed Mutagenesis. Transformed products from 8/18<br />
<br />
Cycles 8 and 9 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/20/10===<br />
Site-Directed Mutagenesis-Redo PCR. 10 colonies were screened, all negative (Two bands were seen when cut with PstI)<br />
<br />
Cycles 10-12 of MAGE for tqsA Knockouts.<br />
----</div>Gcxmatthttp://2010.igem.org/Week11_8/22/10-8/28/10Week11 8/22/10-8/28/102010-10-16T23:06:42Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week11 Highlights===<br />
Finished MAGE cycling and began screening. Assembled Cp-LacPi-RFP combinations for Cp-LacPi characterization. Also continued site directed mutagenesis.<br />
<br />
----<br />
<br />
===8/22/10===<br />
<br />
Cycle 13 of MAGE for tqsA Knockouts.<br />
<br />
----<br />
<br />
===8/23/10===<br />
Site-Directed Mutagenesis<br />
<br />
Cycles 14 and 15 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/24/10===<br />
Ligated CP-LacPI-RFP combinations.<br />
----<br />
<br />
===8/25/10===<br />
<br />
Regrew Cycle 15 MAGE cells in liquid culture.<br />
<br />
----<br />
<br />
===8/26/10===<br />
<br />
Screened MAGE culture for knockouts.<br />
<br />
----<br />
<br />
===8/27/10===<br />
<br />
Screened MAGE culture for knockouts.<br />
<br />
----</div>Gcxmatthttp://2010.igem.org/Week12_8/29/10-9/4/10Week12 8/29/10-9/4/102010-10-16T23:06:35Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week12 Highlights===<br />
We began assembling LacP-RBS-CHS3. We defrosted our -20 and in the process reorganized the -20 and threw out unnecessary DNA.<br />
----<br />
<br />
===8/30/10===<br />
<br />
Screened MAGE culture for knockouts.<br />
<br />
----<br />
<br />
===8/31/10===<br />
Attempted to assemble LacP-RBS-CHS3 --> accidentally cut CHS3 with P (contains a P site)<br />
<br />
Reorganized the racks in the -20. <br />
<br />
Threw out unnecessary/old DNA from our -20.<br />
<br />
Moved all of our racks from our -20 to another -20 in order to defrost our -20.<br />
<br />
tqsA Knockouts FOUND (?) in MAGE culture upon screening.<br />
----<br />
<br />
===9/1/10===<br />
Ragan left for home today and left MAGE in our hands. He asked us to test his tsqA knockouts to determine if they were in fact true knockouts.<br />
<br />
===9/2/10===<br />
***********<br />
----<br />
<br />
===9/3/10===<br />
Reorganized -20 DNA stock. Created racks for individual teammates to stay organized once the school year starts.<br />
----</div>Gcxmatthttp://2010.igem.org/Week10_8/15/10-8/21/10Week10 8/15/10-8/21/102010-10-16T23:06:17Z<p>Gcxmatt: /* Week10 Summary */</p>
<hr />
<div>__NOTOC__<br />
===Week10 Summary===<br />
We continued working on the MAGE tqsA knockouts. We cloned CHS3 into a standard chlor biobricks backbone and began site directed mutagenesis (SDM) to remove the PstI site.<br />
<br />
----<br />
<br />
===8/16/10===<br />
Cloned CHS3 into Chlor Biobricks plasmid --> will be used for Site Directed Mutagenesis<br />
<br />
Cycles 2 and 3 of MAGE for tqsA Knockouts.<br />
<br />
----<br />
<br />
===8/17/10===<br />
Site-Directed Mutagenesis to remove the PstI site in CHS3<br />
<br />
Cycles 4-6 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/18/10===<br />
Site-Directed Mutagenesis. Ran screening for 8/17, none were positive so repeated reaction with better protocol. (PCR only)<br />
<br />
Cycle 7 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/19/10===<br />
Site-Directed Mutagenesis. Transformed products from 8/18<br />
<br />
Cycles 8 and 9 of MAGE for tqsA Knockouts.<br />
----<br />
<br />
===8/20/10===<br />
Site-Directed Mutagenesis-Redo PCR. 10 colonies were screened, all negative (Two bands were seen when cut with PstI)<br />
<br />
Cycles 10-12 of MAGE for tqsA Knockouts.<br />
----</div>Gcxmatthttp://2010.igem.org/Week8_8/1/10-8/7/10Week8 8/1/10-8/7/102010-10-16T23:06:00Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week8 Highlights===<br />
We tried our 3A assemblies using circular plasmid backbones extracted from the iGEM kit instead of the PCRed linear backbone plasmid. This solved our 3A assembly problem and all assemblies worked. We assembled our CP-LacPi inducible promoter parts. We also made competent ChiA knockout cells. We began assembling our (CP-LacPi)-(GFP) constructs to test/characterize the CP-LacPi parts. We assembled pMAL-CHS3 and tried to stain for chitin using calcofluor white.<br />
<br />
----<br />
<br />
===8/2/10===<br />
cP 3A Assembly troubleshooting: why are there white colonies? --> Ran Gel along with Red and Green (lacp-gfp in cpc) <br />
<br />
1050 (rfp default insert), 2150 (bbp), 55+878 (lacp,gfp)<br />
<br />
red should be - 3200, 1050, 2150<br />
<br />
white should be - 2150<br />
<br />
green should be - 3083, 933, 2150<br />
<br />
cP 3A Assembly troubleshooting: How to improve efficiency? Tried Phosphatase in parallel.<br />
<br />
----<br />
<br />
===8/3/10===<br />
Gel Results (White, Green, Red Colonies from 3A Assembly)<br />
*Red colonies look normal<br />
*Green colonies look normal<br />
*White colonies --> abnormal bands at 750bp, 1750bp, 2600bp, and 4300bp<br />
<br />
The white colonies could be due to aggregation of GFP or RFP protein due to excessive amounts of plasmid. <br />
<br />
Inoculated CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2, YdgG Knockout, ChiA Knockout.<br />
----<br />
<br />
===8/4/10===<br />
Miniprepped CP1-LacPi1, CP2-LacPi1, CP3-LacPi1, CP1-LacPi2, CP2-LacPi2, CP3-LacPi2, Holin1-XX, Holin2-XX, PMAL, GFP, LacPi1, LacPi2.<br />
<br />
Miniprepped (CP1-Lacpi1) part --> digested with E/P --> ran a gel <br />
*Should be 35+1372/1370 = 1405/1407 +20(pre/suffix)<br />
<br />
Miniprepped (Holin1-XX) and (Holin2-XX) part --> digested with E/P ran a gel <br />
*Should be 1257, 317 + 129 = 1386, 446 +20 (prefix/suffix) <br />
<br />
Made glycerol stock of ________<br />
----<br />
===8/5/10===<br />
Made competent ChiA knockout cells<br />
<br />
Made electrocompetent ECNR2 cells<br />
<br />
Minipreppred (pMal-CHS3) part<br />
<br />
Assembled and transformed (CP1-LacPI1)-(GFP), (CP2-LacPI1)-(GFP),(CP3-LacPI1)-(GFP),(CP1-LacPI2)-(GFP),(CP2-LacPI2)-(GFP), (CP3-LacPI2)-(GFP)<br />
<br />
----<br />
<br />
===8/6/10===<br />
Lysed and stained (pMal-CHS3) cells with calcofluor white --> for chitin<br />
<br />
Transformed ChiA knockout competent cells with pUC19 --> plan to test competency<br />
----</div>Gcxmatthttp://2010.igem.org/Week5_7/11/10-7/17/10Week5 7/11/10-7/17/102010-10-16T23:05:22Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week5 Highlights===<br />
Our LacP-GFP construct expressed fluorescence. We received a sponsorship from NEB. We also me with Wei Zhang and decided to grow our biofilm on normal agar plates rather than in a flow cell. Our first 3A assembly was successful, but all of our following 3A assemblies failed. We began making competent TOP10 cells. CHS3 was successfully PCRed and purified.<br />
----<br />
<br />
===7/11/10===<br />
Roughly 60% of 1-12O colonies expressed green fluorescence<br />
Only a few colonies of 1-12M expressed green fluorescence<br />
No red fluorescence was seen.<br />
<br />
Miniprepped DNA (LacP1-GFP construct) --> stored in -20°C<br />
<br />
IPTG seems unnecessary for expression of GFP via the LacP promoter - very leaky --> need LacL repressor<br />
<br />
Ran PCR products in gel -> good yield<br />
<br />
Kit to Stock:<br />
<br />
*'''1-18I''' (e0430: YFP) <br />
*'''1-18F''' (e1010: RFP)<br />
*'''1-20L''' (q001121: LacPI1)<br />
*'''1-20P''' (q04121: LacPI2)<br />
*'''1-10H''' (i712019: Luciferase)<br />
*'''1-3A''' (psb1c3: BBPlasmid-C)<br />
*'''1-5A''' (psb1k3: bbplasmid-K)<br />
*'''1-7A''' (psb1T3: bbplasmid-T)<br />
*'''1-1C''' (psb1A3 bbplasmid-A)<br />
<br />
----<br />
<br />
===7/12/10===<br />
<br />
All backbone plasmid transformation colonies appear reddish/purple (due to RFP gene in plasmids)<br />
<br />
Colonies were selected from the yfp (e0430 1-18I), rfp (E1010 1-18F), lacpl1 (Q001121 1-20L), lacpl2 (Q04121 1-20P), luciferase (I712019 1-10H), bbplasmid-c (psb1c3 1-3A), bbplasmid-k (psb1k3 1-5A), bbplasmid-t (psb1T3 1-7A), bbplasmid-A (psb1A3 1-1C), 1-2I, and 2-6K plates and incubated in LB broth at 37C for 16 hours (starting at 6:00pm)<br />
<br />
Received sponsorship from NEB.<br />
<br />
----<br />
<br />
===7/13/10===<br />
Miniprep --> mainly ~200ng/ul (3 parts that we redid were still low = ~50ng/ul)<br />
<br />
Used 3A assembly to assemble LacP1-GFP, LacP2-GFP, and LacPI1-GFP (in 4 different backbone plasmids, psb1A3/psb1T3/psb1C3/psb1K3, made from transforming from well parts) <br />
<br />
Met with Wei Zhang to discuss biofilm growth. Decided flow cells would not be the most cost effective and useful way to grow our E.Coli biofilm. Plan on using normal agar plates. Discovered that BacLight Live/Dead assay can be used on bacteria grown on a plate rather than bacteria growth in broth. We were told to add the BacLight dye to the lawn of bacteria and allow for it to diffuse before washing with ______.<br />
<br />
Received sponsorship from Promega. <br />
----<br />
<br />
===7/14/10===<br />
<br />
None of the assemblies grew.<br />
<br />
'''Group Meeting''':<br />
<br />
*Need to keep notes for future team (i.e. sponsorships, changes to protocol, etc.)<br />
*Made a list of things to get from Promega<br />
<br />
----<br />
<br />
===7/15/10===<br />
PCR Purified CHS3 PCR product<br />
<br />
Ran 3 PCR reactions using Amp, Chl, Tet linearized backbone plasmids as the template<br />
<br />
3A Assembly of CP1-GFP, LacP1-GFP, LacP2-GFP, LacPI1-GFP, LacPI2-GFP, and CHS3-XX. <br />
<br />
Streaked TOP10 cells on agar plates and incubate overnight for making competent cells.<br />
----<br />
<br />
===7/16/10===<br />
None of the assemblies grew.<br />
<br />
PCR Purified backbone plasmid PCR products.<br />
<br />
3A Assembly of CP1-GFP, LacP-GFP, LacPI1-GFP.<br />
<br />
Selected single TOP10 colonies and prepared seed stocks for making competent TOP10 cells.<br />
----</div>Gcxmatthttp://2010.igem.org/Week4_7/4/10-7/10/10Week4 7/4/10-7/10/102010-10-16T23:05:05Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week4 Highlights===<br />
We changed our overnight culture protocol to 13 hours instead of 18 hours, but we still have problems with low DNA yields. We thought maybe our antibiotic levels were to blame. Our first PCR reaction of CHS3 failed. We met with Peggy Saks and learned about the Keio Collection where we can find ChiA and ydgG knockouts. We also purchased our IPTG spray bottles.<br />
<br />
----<br />
<br />
===7/4/10===<br />
13hr(2ml) -> miniprep -> ~45ng/μl<br />
<br />
13hr(3ml) -> miniprep -> ~55ng/μl<br />
<br />
15hr(2ml) -> miniprep -> 40ng/μl<br />
<br />
Also, NB consistently gave higher yields than T10 by 5~10ng/μl<br />
<br />
Note: 18hr gave higher yields than either 13hr or 15hr<br />
<br />
Protocol: 18hr, NB, 5ml incubation (spin all down)<br />
----<br />
<br />
===7/5/10===<br />
CHS3 Gel Extraction -> 8ng/μl<br />
<br />
Restarted CHS3 PCR added 5 cycles.<br />
<br />
Kit to Stock:<br />
<br />
*'''2-8E''' (j06702: RFP)<br />
*'''1-18C''' (j23100: CP1)<br />
*'''1-18K''' (j23104: CP2)<br />
*'''1-18M''' (j23105: CP3)<br />
*'''1-2M''' (b0034: RBS1)<br />
*'''1-2G''' (b0031: RBS2)<br />
*'''1-2I''' (b0032: RBS3)<br />
*'''1-23L'''(b0015: DT1)<br />
*'''1-2O''' (c0012: LacL)<br />
<br />
Reincubated (5μl transfer to liquid culture) 1-12O, 1-12M, 1-6G (GFP1, GFP2, LacP1 respectively)<br />
<br />
Notes: Need Cells; Reorganize Lab (assign benches)<br />
----<br />
<br />
===7/6/10===<br />
Miniprep (16hr/5ml) -> around 50ng/μl<br />
<br />
Ran another gel extraction for CHS3<br />
<br />
Kit to Stock:<br />
*'''1-1D''' (r0010: LacP2)<br />
*'''3-20K''' (K124014: Holin2)<br />
<br />
Plated 1-1D, 3-20K and incubated overnight.<br />
<br />
Colonies were selected from the NB-8E, NB-18C, NB-18K, NB-18M, NB-2M, NB-2G, T10-2I, T10-23L, T10-6K, and T10-2O plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 6:00pm).<br />
----<br />
<br />
===7/7/10===<br />
Miniprep (25ul of Amp/5ml spin down) --> 3 ~100ng/μl and 2 ~180ng/μl <br />
<br />
Miniprep (50ul of Amp/1.5ml spin down) --> all ~20ng/μl<br />
<br />
Miniprep (50ul of Amp/3.5ml spin down) --> all ~20ng/μl<br />
<br />
50μg/ml of Amp = seems to result in more DNA (half the concentration of what we previously used)<br />
<br />
Gel extraction --> very bright CHS3 band (10.1ng/μl)<br />
<br />
Colonies were selected from the T10-2I, T10-23L, T10-6K, T10-2O, T10-1D, T10-20K plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 7:30pm)<br />
<br />
----<br />
<br />
===7/8/10===<br />
Ethanol precipitation of 1-6G, 2-8E, 1-12O, and 1-12M<br />
<br />
Keio Collection: ChiA knockouts grew, but YdgG knockouts did not (possibly dead due to storage in -20 instead of -80)<br />
<br />
Started another PCR reaction of CHS3<br />
<br />
Miniprep (5ml spin down/25ul of antibiotic) --> mostly ~50ng/ul<br />
<br />
Miniprep (5ml spin down/20ul of antibiotic) --> mostly ~70ng/ul<br />
<br />
Diagrammed out our assembly methods for the CHS3 plasmid and the Apoptotic plasmid<br />
<br />
<br />
----<br />
<br />
===7/9/10===<br />
Finished the ethanol precipiation (RESULTS)<br />
<br />
Started assembling the LacP-RFP, LacP-GFP1, and LacP-GFP2 pieces for use in testing the Lac promoter.<br />
<br />
----<br />
===7/10/10===<br />
IPTG Spray Bottles:<br />
10 Sprays = ~1.25ml --> .125ml/spray<br />
100mM IPTG Stpcl = 1 gram/41.96ml<br />
<br />
3 Sprays of 1mM IPTG (started at 5:15pm)<br />
*30 Min<br />
*1 Hour<br />
<br />
3 Sprays of 5mM IPTG (started at 5:15pm)<br />
*30 Min<br />
*1 Hour<br />
----</div>Gcxmatthttp://2010.igem.org/Week3_6/27/10-7/3/10Week3 6/27/10-7/3/102010-10-16T23:04:44Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week3 Highlight===<br />
We re-extracted 12O and 6G and extracted 3-20M. We also met with Dr. Russin about the microscopy aspect of our project. We plan on focusing on confocal microscopy to view the cross section of our biofilm. We established weekly meetings with professor Jewett, Leonard, and Mordacq. We began PCRing CHS3 using yeast genome. We also spoke to Avi about our miniprep problems and he advised us that 18 hours of incubation was "way too long".<br />
<br />
----<br />
<br />
===6/28/10===<br />
Kit to Stock<br />
<br />
*'''1-12O''' (e0840: rbs30-gfp-2xterm)<br />
<br />
*'''1-6G''' (r0011: inducible promoter)<br />
<br />
*'''3-20M''' (K124017: Bacteriophage Lysis Cassette S105, R, and Rz)<br />
<br />
Plates were incubated at 37°C overnight (starting at 2:30 pm).<br />
<br />
----<br />
<br />
===6/29/10===<br />
Selected single 1-12O, 1-6G, 3-20M colonies and grew them in Amp-LB broth for 18 hours (starting at 4:15 pm).<br />
----<br />
<br />
===6/30/10===<br />
<br />
Poured 2 Sleeves of Kan and Amp plates respectively<br />
<br />
Met with Dr. Russin (BIF)<br />
<br />
Sent emails regarding biofilm and biofilm imaging: Aaron Packman, Wei Zhang, Kimberly Grey<br />
<br />
Most likely going to use water immersion Leica (Confocal Microscope) + low MP agar (enrobing technique)<br />
<br />
Red colonies reappeared on 1-12O plates; Fluorescent Microscopy revealed negligible activity; still unsure why colonies are red.<br />
<br />
Miniprepped 1-12O, 3-20M, 1-6G: nanodrop -> mostly below 20ng, 1 above 100ng; stored in -20°C<br />
<br />
Re-miniprepped after growing in liquid LB for 3 hours; nanodrop -> around 50ng/μL, 2 around 100ng/μL; stored in -20°C<br />
<br />
Transferred overnight cultures into new 5ml LB broths. Incubated overnight in an angled shaker (starting from 4:20pm)<br />
<br />
Plated 10μl, 25μl, 100μl, and 200μl (added LB up to 200μL) of the KR 1-12O overnight culture. Intend to measure the thickness of E.Coli lawn.<br />
<br />
----<br />
<br />
===7/1/10===<br />
First weekly meeting with Professor Jewett, Leonard, and Mordacq.<br />
<br />
Removed overnight cultures from incubator at 10:00am. Miniprep (let the elution buffer sit for 10 minutes with the DNA before centrifuging) --> 1 around 30ng/μL, mostly around 75ng/μL.<br />
<br />
Started PCR of the chitin synthase gene using yeast genome.<br />
<br />
Kit to Stock:<br />
*'''1-12O''' (e0840: rbs30-gfp-2xterm)<br />
*'''1-12M''' (e0240: rbs32-gfp-2xterm)<br />
*'''1-6G''' (r0011: inducible promoter)<br />
<br />
Plates were incubated at 37°C overnight (starting at 5pm).<br />
----<br />
<br />
===7/2/10===<br />
Removed overnight plates from 37°C at 10:00am and moved them to the 4°C cold room.<br />
<br />
Gel extraction on Chitin Synthase PCR product.<br />
<br />
Colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 18 hours at 37°C (starting at 4:00pm).<br />
<br />
----<br />
<br />
===7/3/10===<br />
Miniprep -> ~50ng/μl<br />
<br />
Called Avi about kit to stock protocol - "18hr way too long"<br />
<br />
2 Sets of colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 13 and 15 hours at 37°C (starting at 9:15pm). <br />
<br />
----</div>Gcxmatthttp://2010.igem.org/Week2_6/20/10-6/26/10Week2 6/20/10-6/26/102010-10-16T23:04:23Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
<br />
===Week2 Highlights===<br />
We began making antibiotic plates. We also took out parts 12O and 6G from the iGEM kit because we planned on testing inducible promoters. We ran into our first problem when we received low DNA yields after DNA extraction. We also experienced weird reddish/pink colonies in our 12O plates which we expected to be completely white.<br />
<br />
----<br />
<br />
===6/22/10===<br />
<br />
We poured our Kan, Amp, Tet, Kan+Amp, Kan+Tet, Amp+Tet antibiotic agar plates.<br />
<br />
<u>Antibiotic Concentrations</u><br />
*Amp: 50mg/500ml <br />
<br />
*Kan: 31.97mg/500ml<br />
<br />
*Tet: 6mg/500ml<br />
<br />
----<br />
<br />
===6/23/10===<br />
Resuspended 12O and 6G DNA from the iGEM Kit and transformed them into our competent cells.<br />
<br />
*'''6G''' = r0011(inducible promoter)<br />
*'''12O''' = e0840(rbs30-gfp-2xterm)<br />
<br />
----<br />
<br />
===6/24/10===<br />
Ran a gel of our PCR product to make sure that our taq polymerase master mix was working. <br />
<br />
Selected single 12O and 6G colonies and transferred them to LB and incubated for 18 hours.<br />
<br />
Let 1 12O and 1 6G plate incubate at RT. Let 1 12O and 1 6G plate incubate at 37°C. Intended to observe lawn growth.<br />
<br />
----<br />
<br />
===6/25/10===<br />
Low DNA yield; Plan to redo DNA extraction from Kit<br />
<br />
Found red/pink colonies in 12O plates. Thought to be contaminations. Plates were thrown out.<br />
<br />
----</div>Gcxmatthttp://2010.igem.org/Week16_9/26/10-10/2/10Week16 9/26/10-10/2/102010-10-16T23:03:46Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week16 Highlights===<br />
<br />
----<br />
===9/26/10===<br />
Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!<br />
<br />
EDIT THIS:<br />
So the gels I ran today for the digestions didn't turn out. At least no band is less than 1000 bp. Our Cp-Lacpi parts should either be 90 bp for CP(x) - LacPi1 [might be inherently difficult to see] and 235 bp for CP(x) - LacPi2, for reference. Just to do a little troubleshooting, is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid? I'll try to catch Mordacq after my 3 p.m. class to see what he has to say. The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look.<br />
<br />
Also, the bands in the gel were a little wavy. According to Mordacq, this can happen when the agarose is not properly melted all the way. I usually microwave for 1:30. I'll let it go for 30 seconds, then quickly swish it around, and put it back for the remaining 1:00 so it boils all the way. This is more important when not using low melting point agarose.<br />
<br />
Finally, I innoculated the CP-LacPi-GFP parts again today around 7:00. I'll pop them in the centrifuge after sys phys to let them grow for a good amount of time and leave them in there for when you get in lab at 2:00.<br />
<br />
I'll also prepare the two parts for sequencing from the last gel and give them to Mordacq at 3:00.<br />
<br />
Also, here's what we need to get done in the near future...<br />
<br />
- make alkaline miniprep lysis, resuspension and neutralization buffer (need to ask Mordacq where glucose is and how to prepare 1% SDS)<br />
- research plate reader protocols for plate reader exps<br />
- fill out project notebook with what we've been working on<br />
----<br />
<br />
===9/27/10===<br />
Inserted LacP-RBS into Tet plasmid --> transformed into ChiA knockouts<br />
<br />
Kevin went in between classes to spin down the incubations from last night.<br />
<br />
Timi picked up from there and miniprepped the samples and then digested them.<br />
----<br />
<br />
===9/28/10===<br />
pMAL - realized primers are bad, reordering<br />
<br />
Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.<br />
<br />
EDIT:<br />
Still, Kevin wanted to send you the results of the gels.<br />
<br />
Unfortunately, even with digesting more DNA, it is still too difficult to visualize the small inserts. This can be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.<br />
<br />
As far as controlling for DNA run on the gels, we will have to be careful loading the DNA into the gels (given the large volume, there were a few wells where I lost DNA during the transfer) as well as the volumes for our digestions (some of the digestions were not 20 µl -- this can be from liquid evaporating, but since the volumes were inconsistent it can also be pipeting error). Just some things to be aware of.<br />
<br />
In the meantime, we should ask Mordacq what he thinks our next step should be -- whether we want to try again or try another method of screening. We should also double check we're doing the screening process correctly by digesting and running the CP-LACPI-GFP assemblies you just miniprepped. It will be much easier to see the inserts on a gel.<br />
<br />
I'm going to send some things in for sequencing tomorrow morning so I may try to digest before class or from 11 a.m. - 1 p.m. Would you be able to run and image a gel tomorrow later afternoon/evening? Not sure if you have Jumpstart, so just let me know. <br />
----<br />
<br />
===9/29/10===<br />
Site-directed mutagenesis: Amplification and DpnI digestion completed.<br />
<br />
Ragan had some trouble with the TBE buffer. We are looking into what went wrong with it. Sean?<br />
----<br />
<br />
===9/30/10===<br />
Transformation for Site-Directed mutagenesis completed.<br />
<br />
At the lab meeting this morning, Timi discovered that Kevin had been looking for an incorrectly-sized band in the gels. They went back through the photos of gels later in the day, but were inconclusive. They are going to back-track a little bit to figure out if any of the colonies that they had picked were actually positive.<br />
----<br />
<br />
===10/1/10===<br />
The transformation resulted in a good number of colonies for site-directed mutagenesis. 10 liquid cultures were made.<br />
<br />
Timi went in early to pour a big and small gel. BUT she forgot to put in the combs. FAIL.<br />
<br />
Kevin repoured the gels and ran the digested DNA on it. However, he forgot to run the digested parts on it as well, as the advisors had suggested.<br />
----<br />
<br />
===10/2/10===<br />
Timi and Kevin put in 15 overnight inculations of 31-GFP, 32-GFP and 21-GFP.<br />
----</div>Gcxmatthttp://2010.igem.org/Week15_9/19/10-9/25/10Week15 9/19/10-9/25/102010-10-16T23:03:01Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week12 Highlights===<br />
Classes at Northwestern started this week. This complicated our functionality as a team due to scheduling. Thus, we paired up and began working on different portions of the project on our own.<br />
<br />
*Timi/Kevin: CP-LacPI assembly<br />
<br />
*Matt/Sean: Chitin synthase<br />
<br />
*Ben: Site directed mutagenesis<br />
<br />
*Ragan: MAGE<br />
<br />
We submitted our iGEM abstract and has a group meeting for the first time since everyone left to go back home for a few weeks. We realized that something was wrong with our originally PCRed CHS3, so we remade it. However, we were only able to get 25-35 ng/ul of DNA. We also began an extra weekly meeting on Saturdays around 4pm to work on our poster, presentation, and wiki pages.<br />
<br />
----<br />
<br />
===9/20/10===<br />
iGEM abstracts were due tonight. We had an after-hours lab meeting to finish things up. Prof. Leonard stopped by to help us make some final edits and to figure out how much money we had left in the budget.<br />
<br />
We also took this time to catch eachother up on what we had been working on since team members started leaving for home.<br />
----<br />
<br />
===9/21/10===<br />
Timi and Kevin poured a large gel prior to heading home for the night.<br />
----<br />
<br />
===9/22/10===<br />
Timi and Kevin loaded digested CP-LacPI assemblies into gels this morning and Kevin ran the gel between classes.<br />
----<br />
<br />
===9/23/10===<br />
Early 8am team meeting with Prof. Mordacq.<br />
<br />
CHS3 made, low conc (25-35) 5 tubes of about 80ul each in 4degree<br />
----<br />
<br />
===9/24/10===<br />
Digested LacP-RBS(2) with ES.<br />
Digested Tet plasmid with ES.<br />
----<br />
===9/25/10===<br />
We had a group lab meeting to begin working on the poster and presentation.<br />
<br />
Timi and Kevin put 18 overnight cultures of CP-LacPI assemblies into the incubator with Tet antibiotic (UHOH!).<br />
<br />
The Wildcats won their game! 4-0! Go cats! :)<br />
----</div>Gcxmatthttp://2010.igem.org/Week14_9/12/10-9/18/10Week14 9/12/10-9/18/102010-10-16T22:50:04Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week14 Summary===<br />
We ran multiple gels to test our LacP-RBS and LacP-RBS-CHS3 assemblies. We continued preparing and assembling our combinations of Cp-LacPi-GFP for characterization. We also poured more Chlor and Tet plates using smaller (60mm x 15mm) petri dishes given to us by VWR.<br />
----<br />
===9/13/10===<br />
Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.<br />
----<br />
<br />
===9/14/10===<br />
Inoculated Apop1<br />
<br />
Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes<br />
<br />
Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.<br />
----<br />
<br />
===9/15/10===<br />
Miniprepped LacP-RBS-CHS3 and Apop1<br />
<br />
Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)<br />
<br />
Kevin put 15 overnight cultures into the 37 degree incubator at 6pm. <br />
<br />
Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate). <br />
<br />
Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.<br />
----<br />
<br />
===9/16/10===<br />
Ran digested LacP-RBS-CHS3 on a gel:<br />
*Majority contained 1 band around 3600bp<br />
*3 contained 3 bands (2000bp, 1600bp, 1100bp)<br />
<br />
Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.<br />
----<br />
<br />
===9/17/10===<br />
Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel<br />
LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp<br />
<br />
Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.<br />
<br />
Anyhow- results! Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.<br />
<br />
CP3-LacPi2 retrans:<br />
<br />
1) 155.3<br />
<br />
2) 135.1<br />
<br />
3) 76.8<br />
<br />
CP1-LacPi2 seq:<br />
<br />
1) 102.9<br />
<br />
2) 91.7<br />
<br />
3) 71.7<br />
<br />
CP2-LacPi2:<br />
<br />
1) 83.8<br />
<br />
2) 89.6<br />
<br />
CP1-LacPi2<br />
<br />
1) 54.7<br />
<br />
2) 94.7<br />
<br />
CP1-LacPi1:<br />
<br />
1) 108.8<br />
<br />
2) 66.0<br />
<br />
3) 36.5<br />
<br />
The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.<br />
<br />
<br />
PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.<br />
<br />
Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.<br />
----</div>Gcxmatthttp://2010.igem.org/Week14_9/12/10-9/18/10Week14 9/12/10-9/18/102010-10-16T22:46:36Z<p>Gcxmatt: </p>
<hr />
<div>__NOTOC__<br />
===Week14 Summary===<br />
We ran multiple gels to test our LacP-RBS and LacP-RBS-CHS3 assemblies. <br />
----<br />
===9/13/10===<br />
Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.<br />
----<br />
<br />
===9/14/10===<br />
Inoculated Apop1<br />
<br />
Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes<br />
<br />
Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.<br />
----<br />
<br />
===9/15/10===<br />
Miniprepped LacP-RBS-CHS3 and Apop1<br />
<br />
Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)<br />
<br />
Kevin put 15 overnight cultures into the 37 degree incubator at 6pm. <br />
<br />
Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate). <br />
<br />
Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.<br />
----<br />
<br />
===9/16/10===<br />
Ran digested LacP-RBS-CHS3 on a gel:<br />
*Majority contained 1 band around 3600bp<br />
*3 contained 3 bands (2000bp, 1600bp, 1100bp)<br />
<br />
Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.<br />
----<br />
<br />
===9/17/10===<br />
Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel<br />
LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp<br />
<br />
Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.<br />
<br />
Anyhow- results! Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.<br />
<br />
CP3-LacPi2 retrans:<br />
<br />
1) 155.3<br />
<br />
2) 135.1<br />
<br />
3) 76.8<br />
<br />
CP1-LacPi2 seq:<br />
<br />
1) 102.9<br />
<br />
2) 91.7<br />
<br />
3) 71.7<br />
<br />
CP2-LacPi2:<br />
<br />
1) 83.8<br />
<br />
2) 89.6<br />
<br />
CP1-LacPi2<br />
<br />
1) 54.7<br />
<br />
2) 94.7<br />
<br />
CP1-LacPi1:<br />
<br />
1) 108.8<br />
<br />
2) 66.0<br />
<br />
3) 36.5<br />
<br />
The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.<br />
<br />
<br />
PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.<br />
<br />
Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.<br />
----</div>Gcxmatthttp://2010.igem.org/Week14_9/12/10-9/18/10Week14 9/12/10-9/18/102010-10-16T22:45:59Z<p>Gcxmatt: /* 9/17/10 */</p>
<hr />
<div>===9/13/10===<br />
Assembled LacP-RBS-CHS3 using standard assembly. Grew on Chlor plates.<br />
----<br />
<br />
===9/14/10===<br />
Inoculated Apop1<br />
<br />
Inoculated 18 colonies of LacP-RBS-CHS3 for screening purposes<br />
<br />
Plated CHS3,CP-LacPI-GFP, CP-LacPI-RFP.<br />
----<br />
<br />
===9/15/10===<br />
Miniprepped LacP-RBS-CHS3 and Apop1<br />
<br />
Digested LacP-RBS-CHS3 with X and P (CHS3 contains an internal P site)<br />
<br />
Kevin put 15 overnight cultures into the 37 degree incubator at 6pm. <br />
<br />
Kevin the CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assemblies around 9 pm. There was one normal size KAN plate for each CP-LACPI-GFP/RFP assembly (including a negative control) and two small size TET plates for each CP-LACPI assembly (with only one negative control plate). <br />
<br />
Timi poured two sleeves of mini-Chlor and one sleeve of mini-Tet plates.<br />
----<br />
<br />
===9/16/10===<br />
Ran digested LacP-RBS-CHS3 on a gel:<br />
*Majority contained 1 band around 3600bp<br />
*3 contained 3 bands (2000bp, 1600bp, 1100bp)<br />
<br />
Timi mini-prepped the 15 overnight cultures that Kevin put into the incubator last night.<br />
----<br />
<br />
===9/17/10===<br />
Digested LacP-RBS(1), LacP-RBS(2), and LacP-RBS(3) with ES --> Ran on a gel<br />
LacP-RBS(2) and LacP-RBS(3) showed correct bands at ~70bp<br />
<br />
Timi miniprepped the cultures Kevin inoculated last night. Note: the tube caps were snapped shut while they were shaking-- that probably affected the DNA concentration of some of the minipreps.<br />
<br />
'''Results'''<br />
Two of the cultures did not have any cells so he probably just missed the colony or something- not a big deal.<br />
<br />
CP3-LacPi2 retrans:<br />
1) 155.3<br />
<br />
2) 135.1<br />
<br />
3) 76.8<br />
<br />
CP1-LacPi2 seq:<br />
1) 102.9<br />
<br />
2) 91.7<br />
<br />
3) 71.7<br />
<br />
CP2-LacPi2:<br />
1) 83.8<br />
<br />
2) 89.6<br />
<br />
CP1-LacPi2<br />
1) 54.7<br />
<br />
2) 94.7<br />
<br />
CP1-LacPi1:<br />
1) 108.8<br />
<br />
2) 66.0<br />
<br />
3) 36.5<br />
<br />
The concentrations are all on the lower side compared to what we have been getting. I would say to just send some out for sequencing to see if the ligations actually worked before worrying about the concentrations. These DNA vials are in my DNA box in the -20.<br />
<br />
<br />
PLATES:: The CP-LACPI assemblies as well as the CP-LACPI-GFP/RFP assembly plates still had really small colonies on them. Timi came back later to put them into the cold room.<br />
<br />
Kevin put in another series of CP-LacPI-GFP/RFP incubations at around 6pm.<br />
----</div>Gcxmatt