Team:Northwestern/Notebook

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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]
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__NOTOC__
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<html>
<html>
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<head>
 
<style>
<style>
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body {
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/* Wiki Hacks - START */
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  background: white url(https://static.igem.org/mediawiki/2010/3/38/NUbackground.jpg) repeat-x;
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#globalWrapper {
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}
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    background-color: transparent;
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    padding-bottom:0px;
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</head>
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    border: none;
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</html>
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    }
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<!--- The Mission, Experiments --->
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#top-section {
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{|- style="background:#FFFFFF; style="font-size: 87%;" color:#000000" cellpadding="1" cellspacing="1" bordercolor="#000000" width="97%" align="center"
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    height: 0px;
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!align="center"|[[Team:Northwestern|<span style="color:#000000;">'''Home'''</span>]]
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    margin-top: 0px;
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!align="center"|[[Team:Northwestern/Brainstorm|<span style="color:#000000;">'''Brainstorm'''</span>]]
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    margin-left: auto;
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!align="center"|[[Team:Northwestern/Team|<span style="color:#000000;">'''Team'''</span>]]
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    margin-right: auto;
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!align="center"|[[Team:Northwestern/Acknowledgements|<span style="color:#000000;">'''Acknowledgements'''</span>]]
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    margin-bottom: 0 !important;
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!align="center"|[[Team:Northwestern/Project|<span style="color:#000000;">'''Project'''</span>]]
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    margin-bottom: 0px;
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!align="center"|[[Team:Northwestern/SideProject|<span style="color:#000000;">'''Side Project'''</span>]]
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    padding:0;
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!align="center"|[[Team:Northwestern/Parts|<span style="color:#000000;">'''Parts'''</span>]]
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    border: 1;
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!align="center"|[[Team:Northwestern/Notebook|<span style="color:#000000;">'''Notebook'''</span>]]
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    }
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!align="center"|[[Team:Northwestern/Calendar|<span style="color:#000000;">'''Calendar'''</span>]]
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#p-logo {
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!align="center"|[[Team:Northwestern/Protocol|<span style="color:#000000;">'''Protocol'''</span>]]
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    height:0px;
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!align="center"|[[Team:Northwestern/Safety|<span style="color:#000000;">'''Safety'''</span>]]
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    overflow:hidden;
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!align="center"|[[Team:Northwestern/Links|<span style="color:#000000;">'''Links'''</span>]]
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    border:none;
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!align="center"|[[Team:Northwestern/References|<span style="color:#000000;">'''References'''</span>]]
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    }
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!align="center"|[[Team:Northwestern/Media|<span style="color:#000000;">'''Media'''</span>]]
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#search-controls {
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!align="center"|[[Team:Northwestern/Contact|<span style="color:#000000;">'''Contact'''</span>]]
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    overflow:hidden;
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|}
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    }          
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===6/14-18/10 (Boot Camp)===
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/* Wiki Hacks - END */
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DNA extracted from kit
 
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Part: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_I746200]
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#content {
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    background-color: transparent;
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    width:99%;
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    position: relative;
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    float: center;
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}
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----
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#menubar {
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background-color:transparent;
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font-size:85%;
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line-height:1em;
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position:absolute;
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top:10px;
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white-space:nowrap;
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width:50%;
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z-index:5;
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height:25px;
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overflow:hidden;
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}
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===6/22/10===
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#menubar li a {
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color:#000000;
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}
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We poured our Kan, Amp, Tet, Kan+Amp, Kan+Tet, Amp+Tet antibiotic agar plates.
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.left-menu {
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left:0;
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padding-left:13px;
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}
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.right-menu  {
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right:0;
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}
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<u>Antibiotic Concentrations</u>
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.firstHeading { display:none; }
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*Amp: 50mg/500ml
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}
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*Kan: 31.97mg/500ml
 
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*Tet: 6mg/500ml
 
-
----
 
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===6/23/10===
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#footer-box {
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Resuspended 12O and 6G DNA from the iGEM Kit and transformed them into our competent cells.
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background-color: transparent;
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color: #000000;
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border: 0px;
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margin:0 auto;
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padding:13px 5px;
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width:auto;
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margin-top:-1px;
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}
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*'''6G''' = r0011(inducible promoter)
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#footer-box a {
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*'''12O''' = e0840(rbs30-gfp-2xterm)
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        background-color: transparent;
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color: #000000;
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font-size:90%;
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----
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}
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/* Wiki Hacks - END */
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</style>
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</html>
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===6/24/10===
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<html>
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Ran a gel of our PCR product to make sure that our taq polymerase master mix was working.  
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<head>
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<style>
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body {
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  background: #EFCDF8; <!-- EEDCC8 url(https://static.igem.org/mediawiki/2010/3/38/BG1.jpg) repeat-y; -->
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}
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</style>
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</head>
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</html>
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Selected single 12O and 6G colonies and transferred them to LB and incubated for 18 hours.
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[[Image:LOGOBANNER1.jpg|936px|center|link=http://wikipedia.org|Tech Institute]]
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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.
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<!--- The Mission, Experiments --->
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{|- style="font-size: 87%; background: #EFCDF8; color: #FFFFFF" cellpadding="1" cellspacing="1" bordercolor="#000000" width="75%" align="center"
 +
!align="center"|[[Team:Northwestern|<font color="#2B3856">'''Home'''</font>]]
 +
!align="center"|[[Team:Northwestern/Brainstorm|<font color="#000000">'''Brainstorm'''</font>]]
 +
!align="center"|[[Team:Northwestern/Team|<font color="#000000">'''Team'''</font>]]
 +
!align="center"|[[Team:Northwestern/Acknowledgements|<font color="#000000">'''Acknowledgements'''</font>]]
 +
!align="center"|[[Team:Northwestern/Project|<font color="#000000">'''Project'''</font>]]
 +
!align="center"|[[Team:Northwestern/SideProject|<font color="#000000">'''Human Practices'''</font>]]
 +
!align="center"|[[Team:Northwestern/Parts|<font color="#000000">'''Parts'''</font>]]
 +
!align="center"|[[Team:Northwestern/Notebook|<font color="#000000">'''Notebook'''</font>]]
 +
!align="center"|[[Team:Northwestern/Calendar|<font color="#000000">'''Calendar'''</font>]]
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!align="center"|[[Team:Northwestern/Protocol|<font color="#000000">'''Protocol'''</font>]]
 +
!align="center"|[[Team:Northwestern/Safety|<font color="#000000">'''Safety'''</font>]]
 +
!align="center"|[[Team:Northwestern/Links|<font color="#000000">'''Links'''</font>]]
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!align="center"|[[Team:Northwestern/References|<font color="#000000">'''References'''</font>]]
 +
!align="center"|[[Team:Northwestern/Media|<font color="#000000">'''Media'''</font>]]
 +
!align="center"|[[Team:Northwestern/Contact|<font color="#000000">'''Contact'''</font>]]
 +
|}
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----
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{| align="center" border="0" width="75%"
 +
|
 +
='''Notebook'''=
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===6/25/10===
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[[Brainstorming April-June 2010]]
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Low DNA yield; Plan to redo DNA extraction from Kit
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-
Found red/pink colonies in 12O plates. Thought to be contaminations. Plates were thrown out.
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NOTE: All work done by the undergraduate students of NU iGEM.
 +
=='''June'''==
 +
[[Week1 6/13/10-6/19/10]]
-
----
+
[[Week2 6/20/10-6/26/10]]
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===6/28/10===
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[[Week3 6/27/10-7/3/10]]
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Kit to Stock
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*'''1-12O''' (e0840: rbs30-gfp-2xterm)
 
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*'''1-6G''' (r0011: inducible promoter)
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=='''July'''==
 +
[[Week4 7/4/10-7/10/10]]
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*'''3-20M''' (K124017: Bacteriophage Lysis Cassette S105, R, and Rz)
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[[Week5 7/11/10-7/17/10]]
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Plates were incubated at 37°C overnight (starting at 2:30 pm).
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[[Week6 7/18/10-7/24/10]]
-
----
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[[Week7 7/25/10-7/31/10]]
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===6/29/10===
 
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Selected single 1-12O, 1-6G, 3-20M colonies and grew them in Amp-LB broth for 18 hours (starting at 4:15 pm).
 
-
----
 
-
===6/30/10===
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=='''August'''==
 +
[[Week8 8/1/10-8/7/10]]
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Poured 2 Sleeves of Kan and Amp plates respectively
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[[Week9 8/8/10-8/14/10]]
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Met with Dr. Russin (BIF)
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[[Week10 8/15/10-8/21/10]]
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Sent emails regarding biofilm and biofilm imaging: Aaron Packman, Wei Zhang, Kimberly Grey
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[[Week11 8/22/10-8/28/10]]
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Most likely going to use water immersion Leica (Confocal Microscope) + low MP agar (enrobing technique)
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[[Week12 8/29/10-9/4/10]]
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Red colonies reappeared on 1-12O plates; Fluorescent Microscopy revealed negligible activity; still unsure why colonies are red.
 
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Miniprepped 1-12O, 3-20M, 1-6G: nanodrop -> mostly below 20ng, 1 above 100ng; stored in -20°C
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=='''September'''==
 +
[[Week13 9/5/10-9/11/10]]
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Re-miniprepped after growing in liquid LB for 3 hours; nanodrop -> around 50ng/μL, 2 around 100ng/μL; stored in -20°C
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[[Week14 9/12/10-9/18/10]]
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Transferred overnight cultures into new 5ml LB broths. Incubated overnight in an angled shaker (starting from 4:20pm)
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[[Week15 9/19/10-9/25/10]]
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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.
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[[Week16 9/26/10-10/2/10]]
-
----
 
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===7/1/10===
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=='''October'''==
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First weekly meeting with Professor Jewett, Leonard, and Mordacq.
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[[Week17 10/3/10-10/9/10]]
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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.
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[[Week18 10/10/10-10/16/10]]
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Started PCR of the chitin synthase gene using yeast genome.
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[[Week19 10/17/10-10/23/10]]
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Kit to Stock:
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[[Week20 10/24/10-10/30/10]]
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*'''1-12O''' (e0840: rbs30-gfp-2xterm)
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*'''1-12M''' (e0240: rbs32-gfp-2xterm)
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*'''1-6G''' (r0011: inducible promoter)
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Plates were incubated at 37°C overnight (starting at 5pm).
 
-
----
 
-
===7/2/10===
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=='''November'''==
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Removed overnight plates from 37°C at 10:00am and moved them to the 4°C cold room.
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[[Jamboree at MIT 11/05/10-11/07/10 :: Team Perspectives]]
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Gel extraction on Chitin Synthase PCR product.
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|}
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-
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).
+
-
 
+
-
----
+
-
 
+
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===7/3/10===
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Miniprep -> ~50ng/μl
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-
 
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Called Avi about kit to stock protocol - "18hr way too long"
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-
 
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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).
+
-
 
+
-
----
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-
 
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===7/4/10===
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13hr(2ml) -> miniprep -> ~45ng/μl
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-
 
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13hr(3ml) -> miniprep -> ~55ng/μl
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-
 
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15hr(2ml) -> miniprep -> 40ng/μl
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Also, NB consistently gave higher yields than T10 by 5~10ng/μl
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-
 
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Note: 18hr gave higher yields than either 13hr or 15hr
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Protocol: 18hr, NB, 5ml incubation (spin all down)
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-
----
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-
 
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===7/5/10===
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CHS3 Gel Extraction -> 8ng/μl
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Restarted CHS3 PCR added 5 cycles.
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Kit to Stock:
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*'''2-8E''' (j06702: RFP)
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*'''1-18C''' (j23100: CP1)
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*'''1-18K''' (j23104: CP2)
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*'''1-18M''' (j23105: CP3)
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*'''1-2M''' (b0034: RBS1)
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*'''1-2G''' (b0031: RBS2)
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*'''1-2I''' (b0032: RBS3)
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*'''1-23L'''(b0015: DT1)
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*'''1-2O''' (c0012: LacL)
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Reincubated (5μl transfer to liquid culture) 1-12O, 1-12M, 1-6G (GFP1, GFP2, LacP1 respectively)
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Notes: Need Cells; Reorganize Lab (assign benches)
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-
----
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===7/6/10===
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Miniprep (16hr/5ml) -> around 50ng/μl
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Ran another gel extraction for CHS3
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Kit to Stock:
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*'''1-1D''' (r0010: LacP2)
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*'''3-20K''' (K124014: Holin2)
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Plated 1-1D, 3-20K and incubated overnight.
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-
 
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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).
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-
----
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-
 
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===7/7/10===
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Miniprep (25ul of Amp/5ml spin down) --> 3 ~100ng/μl and 2 ~180ng/μl
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-
 
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Miniprep (50ul of Amp/1.5ml spin down) --> all ~20ng/μl
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-
 
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Miniprep (50ul of Amp/3.5ml spin down) --> all ~20ng/μl
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-
 
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50μg/ml of Amp = seems to result in more DNA (half the concentration of what we previously used)
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-
 
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Gel extraction --> very bright CHS3 band (10.1ng/μl)
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-
 
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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)
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-
 
+
-
----
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-
 
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===7/8/10===
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Ethanol precipitation of 1-6G, 2-8E, 1-12O, and 1-12M
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Keio Collection: ChiA knockouts grew, but YdgG knockouts did not (possibly dead due to storage in -20 instead of -80)
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-
 
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Started another PCR reaction of CHS3
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-
 
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Miniprep (5ml spin down/25ul of antibiotic) --> mostly ~50ng/ul
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Miniprep (5ml spin down/20ul of antibiotic) --> mostly ~70ng/ul
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Diagrammed out our assembly methods for the CHS3 plasmid and the Apoptotic plasmid
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-
 
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-
 
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----
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===7/9/10===
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Finished the ethanol precipiation (RESULTS)
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-
 
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Started assembling the LacP-RFP, LacP-GFP1, and LacP-GFP2 pieces for use in testing the Lac promoter.
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-
 
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-
----
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===7/10/10===
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IPTG Spray Bottles:
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10 Sprays = ~1.25ml --> .125ml/spray
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100mM IPTG Stpcl = 1 gram/41.96ml
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-
 
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3 Sprays of 1mM IPTG (started at 5:15pm)
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*30 Min
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*1 Hour
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3 Sprays of 5mM IPTG (started at 5:15pm)
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*30 Min
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*1 Hour
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-
----
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-
 
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===7/11/10===
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roughly 60% of 1-12O colonies express gfp
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only a few colonies of 1-12M express gfp
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rfp not expressed
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-
 
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miniprepped DNA (lac-gfp(12O) construct) in -20
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-
 
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iptg seems irrelevant to expression of gfp - very leaky lacp, esp in T10 -> need lacl
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-
 
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ran pcr products in gel -> good yield
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-
 
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kit to stock:
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-
 
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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)
+
-
 
+
-
----
+
-
 
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-
===7/12/10===
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-
 
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All backbone plasmid transformation colonies appear reddish/purple (due to RFP gene in plasmids)
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-
 
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-
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)
+
-
 
+
-
----
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-
 
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===7/13/10===
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-
----
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===7/14/10===
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-
----
+
-
===7/15/10===
+
-
----
+
-
===7/16/10===
+

Latest revision as of 05:54, 20 March 2011


Tech Institute
Home Brainstorm Team Acknowledgements Project Human Practices Parts Notebook Calendar Protocol Safety Links References Media Contact

Notebook

Brainstorming April-June 2010

NOTE: All work done by the undergraduate students of NU iGEM.

June

Week1 6/13/10-6/19/10

Week2 6/20/10-6/26/10

Week3 6/27/10-7/3/10


July

Week4 7/4/10-7/10/10

Week5 7/11/10-7/17/10

Week6 7/18/10-7/24/10

Week7 7/25/10-7/31/10


August

Week8 8/1/10-8/7/10

Week9 8/8/10-8/14/10

Week10 8/15/10-8/21/10

Week11 8/22/10-8/28/10

Week12 8/29/10-9/4/10


September

Week13 9/5/10-9/11/10

Week14 9/12/10-9/18/10

Week15 9/19/10-9/25/10

Week16 9/26/10-10/2/10


October

Week17 10/3/10-10/9/10

Week18 10/10/10-10/16/10

Week19 10/17/10-10/23/10

Week20 10/24/10-10/30/10


November

Jamboree at MIT 11/05/10-11/07/10 :: Team Perspectives

Retrieved from "http://2010.igem.org/Team:Northwestern/Notebook"