Team:Columbia-Cooper-NYC/Columbia notebook 2

From 2012.igem.org

(Difference between revisions)
(Monday, 30th)
Line 1: Line 1:
 +
<!--- The Mission, Experiments --->
 +
<!--
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
!align="center"|[[Team:Columbia-Cooper-NYC|Home]]
!align="center"|[[Team:Columbia-Cooper-NYC|Home]]
Line 10: Line 12:
!align="center"|[[Team:Columbia-Cooper-NYC/Attributions|Attributions]]
!align="center"|[[Team:Columbia-Cooper-NYC/Attributions|Attributions]]
!align="center"|[[Team:Columbia-Cooper-NYC/Sponsor Us|Sponsor Us]]
!align="center"|[[Team:Columbia-Cooper-NYC/Sponsor Us|Sponsor Us]]
 +
!align="center"|[[Team:Columbia-Cooper-NYC/Trial|Trial]]
|}
|}
 +
-->
 +
 +
<html>
 +
<head>
 +
<body>
 +
<!-- dd menu -->
 +
<script type="text/javascript">
 +
<!--
 +
var timeout        = 500;
 +
var closetimer = 0;
 +
var ddmenuitem      = 0;
 +
 +
// open hidden layer
 +
function mopen(id)
 +
{
 +
// cancel close timer
 +
mcancelclosetime();
 +
 +
// close old layer
 +
if(ddmenuitem) ddmenuitem.style.visibility = 'hidden';
 +
 +
// get new layer and show it
 +
ddmenuitem = document.getElementById(id);
 +
ddmenuitem.style.visibility = 'visible';
 +
 +
}
 +
// close showed layer
 +
function mclose()
 +
{
 +
if(ddmenuitem) ddmenuitem.style.visibility = 'hidden';
 +
}
 +
 +
// go close timer
 +
function mclosetime()
 +
{
 +
closetimer = window.setTimeout(mclose, timeout);
 +
}
 +
 +
// cancel close timer
 +
function mcancelclosetime()
 +
{
 +
if(closetimer)
 +
{
 +
window.clearTimeout(closetimer);
 +
closetimer = null;
 +
}
 +
}
 +
 +
// close layer when click-out
 +
document.onclick = mclose;
 +
// -->
 +
</script>
 +
<style>
 +
#sddm
 +
{ margin: 0;
 +
padding: 0;
 +
      z-index: 30}
 +
 +
#sddm li
 +
{ margin: 0;
 +
padding: 0;
 +
list-style: none;
 +
float: left;
 +
font: bold 11px arial}
 +
 +
#sddm li a
 +
{ display: block;
 +
margin: 0 1px 0 0;
 +
padding: 4px 10px;
 +
width: 85px;
 +
background: #238E23;
 +
color: #FFF;
 +
text-align: center;
 +
text-decoration: none}
 +
 +
#sddm li a:hover
 +
{ background: #B87333}
 +
 +
#sddm div
 +
{ position: absolute;
 +
visibility: hidden;
 +
margin: 0;
 +
padding: 0;
 +
background: #4CC552;
 +
border: 1px solid #5C3317}
 +
 +
#sddm div a
 +
{ position: relative;
 +
display: block;
 +
margin: 0;
 +
padding: 5px 10px;
 +
width: auto;
 +
white-space: nowrap;
 +
text-align: left;
 +
text-decoration: none;
 +
background: #B5EAAA;
 +
color: #5C3317;
 +
font: 11px arial}
 +
 +
#sddm div a:hover
 +
{ background: #49A3FF;
 +
color: #FFF}
 +
</style>
 +
 +
 +
<!-- div class="sample" style="margin-bottom: 15px;height:42px;"><span -->
 +
<ul id="sddm" style="width:1000px;margin:0 auto">
 +
<li><a href="http://2012.igem.org/Team:Columbia-Cooper-NYC" onmouseover="mopen('m1')" onmouseout="mclosetime()">Home</a>
 +
<div id="m1" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
</div>
 +
</li>
 +
<li><a href="#" onmouseover="mopen('m2')" onmouseout="mclosetime()">Team</a>
 +
<div id="m2" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
<a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Team">Biographies</a>
 +
<a href="#">Facilitators</a>
 +
<a href="#">The University(s)</a>
 +
<a href="#">Official Profile</a>
 +
</div>
 +
</li>
 +
<li><a href="#" onmouseover="mopen('m3')" onmouseout="mclosetime()">Project</a>
 +
<div id="m3" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
<a href="#">Overview</a>
 +
<a href="#">The Problem</a>
 +
                <a href="#">Promoters</a>
 +
                <a href="#">Data</a>
 +
                <a href="#">Modeling</a>
 +
                <a href="#">Conclusions/Accomplishments</a>
 +
                <a href="#">Future Directions</a>
 +
                <a href="#">References</a>
 +
</div>
 +
</li>
 +
<li><a href="#" onmouseover="mopen('m4')" onmouseout="mclosetime()">Parts</a>
 +
<div id="m4" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
<a href="#">Parts Submitted</a>
 +
<a href="#">Attributions</a>
 +
</div>
 +
</li>
 +
<li><a href="#" onmouseover="mopen('m5')" onmouseout="mclosetime()">Lab Notebook</a>
 +
<div id="m5" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
<a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Columbia_notebook_2">Genetics</a>
 +
<a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Columbia_notebook_1">Cooper Etching</a>
 +
<a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Protocols">Protocols</a>
 +
</div>
 +
</li>
 +
        <li><a href="#" onmouseover="mopen('m6')" onmouseout="mclosetime()">Human Practice</a>
 +
<div id="m6" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
<a href="#">Outreach</a>
 +
<a href="#">Collaboration</a>
 +
</div>
 +
</li>
 +
        <li><a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Safety" onmouseover="mopen('m7')" onmouseout="mclosetime()">Safety</a>
 +
<div id="m7" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
                <a href="#">MSDS Sheets</a>
 +
</div>
 +
</li>
 +
        <li><a href="#" onmouseover="mopen('m8')" onmouseout="mclosetime()">Sponsors</a>
 +
<div id="m8" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
                <a href="http://2012.igem.org/Team:Columbia-Cooper-NYC/Sponsor_Us">Sponsor Us</a>
 +
<a href="#">Sponsors</a>
 +
<a href="#">Acknowledgements</a>
 +
</div>
 +
</li>
 +
        <li><a href="#" onmouseover="mopen('m9')" onmouseout="mclosetime()">iGEM</a>
 +
<div id="m9" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">
 +
</div>
 +
</li>
 +
</ul>
 +
<div style="clear:both"></div>
 +
<!-- /span></div -->
 +
<!-- /dd -->
 +
</script>
 +
</body>
 +
</head>
 +
</html>
 +
 +
 +
<!--
 +
<head>
 +
<style>
 +
h2{
 +
font: 1.5em 'Myriad Pro', 'Gill Sans', 'Arial', sans-serif;
 +
font-weight: bold;
 +
color: black;
 +
}
 +
 +
p{
 +
font: 1.2em Georgia, serif;
 +
color: black;
 +
}
 +
 +
#fredbuilding{
 +
width: 500px;
 +
margin-left: auto;
 +
margin-right: auto;
 +
}
 +
</style>
 +
</head>
 +
-->
  __NOTOC__  
  __NOTOC__  

Revision as of 02:54, 8 September 2012




Columbia Genetics Lab Notebook

July, 2012

Week 1

Thursday, 5th

  • Re-hydrated plasmids with 50µl of LB and Kanamycin solution
  • Stored solution at 37°C incubator overnight

Friday, 6th

  • Purified pET26b vector using standard DNA purification protocol

Week 2

Monday, 9th

  • Received kill gene Bba-K124017 from plate 3, 20M
  • Re-hydrated DNA according to standard iGEM re-hydration protocol

Tuesday, 10th

  • Contacted professors at Germany in hopes to receive copies of fungal phytochrome FphA

Wednesday, 11th

  • Received confirmation by professors at Germany for FphA to be sent to Columbia University
  • Conducted transformation using electroporation with competent bacteria (marked by resistance to Kanamycin)
    1. Control: 1µl of deionized water with abt. and 60µl of bacteria cells
    2. Variable: 1µl of re-hydrated kill gene and 60µl of bacteria cells
  • Placed both samples after electroporation into 200µl of preprepared LB
  • Placed samples in shaker 37°C for 30 minutes

Thursday, 12th

  • Grew 1 colony of transformed bacteria in 5mL of Kanamycin and LB solution
    • Note: Using pET20b vector over pET26b vector from glycerol stock solution

Friday, 13th

  • Isolated 4 samples of plasmid using standard plasmid isolation protocol
    1. 2 samples: kill gene
    2. 2 samples: pET20b vector

Week 3

Monday, 16th

  • Re-hydrated two biobrick parts in plasmid pSB2K3 according to standard iGEM re-hydration protocol
    1. BBa-I16009 (PcyA) from plate 1, 20F
    2. BBa-I16008 (ho1) from plate 2, 13J
  • Electroporated 1µl of each biobrick into separate E. coli at 1800V
  • Added 100µl LB broth into each sample
  • Placed samples at 33.4°C for 20 minutes
  • Samples were plated to be grown overnight

Tuesday, 17th

  • Placed 5ml each of LB/Kan into two centrifuge tube for PCB creation
    1. Label P: PcyA
    2. Label h: ho1
  • Placed samples in 37°C incubator

Wednesday, 18th

  • Purified ho1 and PcyA plasmids using standard DNA purification protocol
  • Placed purified DNA into glycerol stock (LB/Kan) and stored at -80°C

Thursday, 19th

  • Purified GFP using standard DNA purification protocol
  • Prepared glycerol stock solution (500µl GFP/500µl 80% glycerol) and stored at -80°C

Week 4

Tuesday, 24th

  • Re-hydrated four biobrick parts according to standard iGEM re-hydration protocol
    1. Inducible plasmid (pSB1AK3-J04500) from plate 4, 12A
    2. GFP (pSB1A2-E0040) from plate 1, 14K
    3. High copy plasmid pSB1T3-J044500 from plate 1, 7A
    4. Low copy plasmid (pSB3C5-J044500) from plate 1, 3C
  • Electroporated competent E. coli with each of the four above genes separetely
  • Created Kan, Amp, Cam, Tetra, and Amp/Kan plates

Wednesday, 25th

  • Streaked pSB1T3-J04450
  • Created LB solution with Kan or Amp or Cam

Thursday, 26th

  • Prepared Glycerol stock for inducible promoter, GFP, and low-copy plasmid
  • Picked a single colony from pSB1T3-J04450 and let it grow overnight in 37C
  • Recorded and measured the DNA concentrations of following at 260nm:
    1. Kill gene
    2. PcyA
    3. ho1
    4. GFP
    5. Inducible promoter
    6. Low copy CAM plasmid
  • Followed digestion and ligation protocol; setup explained below:
    1. Upstream: ho1; Downstream: PcyA; Destination plasmid: Low-copy CAM
    2. Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low-copy CAM
    3. Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: High-copy TET (pSB1T3)
  • After completion, store samples at -20C

Friday, 27th

  • Electroporated ligated samples from previous day: GFP, PCB, Kill
  • Followed standard plasmid isolation protocol for pSB1T3-J04450 (TET plasmid)

Saturday, 28th

  • Check plates from electroporation from previous day

Week 5

Monday, 30th

  • Stored pif3 and phyB that arrived from Sweden
  • Relocated and reorganized the iGEM biobrick kit and glycerol stocks
  • Picked colonies for following DNA:
    1. Inducible promotor (pSB1AK3_J04500)
    2. GFP (pSB1A2_E0040)
    3. Low copy CAM plasmid (pSB3C5_J04150)
  • Added 10µl of ho1 and PcyA to 5ml of antibiotics

August, 2012

Week 5

Week 6

Saturday, 11th

  • Reviewed the solutions for diluted GFP and observed no significant results
  • Reorganized samples in fridges and incubators

Week 7

Monday, 13th

  • Chemically transformed competent cells (BL21) with plasmids below using bioline protocol (used 1/2 of recommended amount)
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
    4. CAM control plasmid
    5. PUC19 control plasmid
  • Electroporated competent cells (α-select) with plasmids below using bioline protocol
    1. Upps 4-Kill gene-High Copy (TET)
    2. Upps 4-Upps 5-High Copy (TET)
    3. Upps 4-Upps 6-High Copy (TET)
    4. PUC19 control plasmid
    5. High copy TET control plasmid

Note 1: TET control sparked

Note 2: Upps 4-Upps 5-TET sparked

Note 3: Original DNA for Upps 4-Upps 5-TET was pink

  • Placed transformed samples in growth media and placed in 37°C shaker
  • Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
    • First row
      1. 2 log ladder
      2. Upps 4
      3. blank
      4. High Copy TET plasmid
      5. Upps 4 (2)
      6. Upps 6
      7. High Copy TET plasmid (2)
      8. Upps 4 (3)
      9. Kill gene
      10. High Copy TET plasmid (3)
      11. PCB
      12. High Copy TET plasmid (4)
    • Second row
      1. 2 log ladder
      2. Inducible promoter-IPTG
      3. Kill gene (2)
      4. High Copy TET plasmid (5)
      5. Inducible promoter-IPTG (2)
      6. Upps 6 (2)
      7. High Copy TET plasmid (6)
      8. Inducible promoter-IPTG (3)
      9. Upps 5 (2)
      10. High Copy TET plasmid (7)
  • Ran gel for 25 minutes at constant 150V
  • Took picture under UV light
  • Created TET and CAM plates

Wednesday, 15th

  • Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
  • Purified above plasmids and CAM control plasmid using standard purification protocol and prepared glycerol stocks
  • Added appropriate buffers to IPTG-Upps 5 and IPTG-Upps 6 and centrifuged for 10 minutes to determine for a pellet
  • Measured OD 600 of following samples
    1. IPTG-Upps 5-Low Copy (CAM): .160A
    2. IPTG-Upps 6-Low Copy (CAM): .038A
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight

Thursday, 16th

  • Measured OD 600 for 200x diluted bacterial solution containing plasmids with promoter inducible with IPTG
    1. IPTG-Upps 5-Low Copy (CAM): .040A
    2. IPTG-Upps 6-Low Copy (CAM): .032A
    3. IPTG-Kill gene-Low Copy (CAM): .028A
  • Noted that cell concentration was dense, decided to dilute solution with 75µl cells and 925µl LB/CAM solution
  • Placed diluted cell solution into 37°C incubator for 40 minutes
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight