Team:Washington/Protocols/VectorAssay

From 2010.igem.org

(Difference between revisions)
(New page: create)
Line 1: Line 1:
-
create
+
{{Template:Team:Washington/Templates/Header}}
 +
<html>
 +
<script type="text/javascript">
 +
$(function() {
 +
  var mainimg = document.getElementById("mainimg");
 +
  var default_src = mainimg.src;
 +
  var restore_mainimg = function() {
 +
    mainimg.src = default_src;
 +
  };
 +
  $(document.getElementById("area_target")).hover(function() {
 +
    mainimg.src = "/wiki/images/1/1d/Main_graphic2_target.png";
 +
  }, restore_mainimg);
 +
  $(document.getElementById("area_secretion")).hover(function() {
 +
    mainimg.src = "/wiki/images/2/23/Main_graphic2_secretion.png";
 +
  }, restore_mainimg);
 +
  $(document.getElementById("area_display")).hover(function() {
 +
    mainimg.src = "/wiki/images/a/aa/Main_graphic2_display.png";
 +
  }, restore_mainimg);
 +
  $(document.getElementById("area_release")).hover(function() {
 +
    mainimg.src = "/wiki/images/e/ed/Main_graphic2_release.png";
 +
  }, restore_mainimg);
 +
});
 +
</script>
 +
</html>
 +
<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
 +
== GFP expression assay ==
 +
 
 +
'''Day 1:  OVERNIGHTS'''
 +
 
 +
*Prepare a 96 deep well plate with 1ml of TB + antibiotic
 +
*Inoculate well by taking at scraping from glycerol stock
 +
*Shake at 37deg 16-24hrs
 +
 
 +
'''Day 2: EXPRESSION'''
 +
 
 +
*Prepare 96 deep well plate with 1ml of TB + antibiotic,
 +
*Inoculate well by taking 20ul of the overnight and place it in 1ml TB, do all inducible twice so to allow for induced verse uninducible expression
 +
*Grow on shaker at 37C for 1 hour
 +
*Induce all inducible constructs with 50ml of 10mM IPTG
 +
*Allow to grow for 18 hour at room temperature on a shaker
 +
 
 +
'''Day 3: DATA'''
 +
 
 +
*Take overnights plates and spin on plate centrifuge for 20 minutes at 4000rpm
 +
*Pour of broth and resuspend in 1ml PBS 7.5 pH on plate shaker
 +
*Spin down PBS suspension for 20 minutes at 4000rpm
 +
*Pour of supernatant
 +
*Resuspend in 1ml PBS 7.5 pH on plate shaker
 +
*Take 100ul of PBS suspension and place into clear bottom plate reader plate
 +
*Take 100ul of PBS suspension and place into black plate reader plate
 +
*Read plates at 525nm and record data
 +
 
 +
 
 +
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
 +
<div style="text-align:center">
 +
'''&larr; [[Team:Washington/Protocols|Back to Lab Protocols]]'''
 +
&nbsp; &nbsp; &nbsp;
 +
</div>
 +
 
 +
{{Template:Team:Washington/Templates/Footer}}

Revision as of 03:34, 23 October 2010

GFP expression assay

Day 1: OVERNIGHTS

  • Prepare a 96 deep well plate with 1ml of TB + antibiotic
  • Inoculate well by taking at scraping from glycerol stock
  • Shake at 37deg 16-24hrs

Day 2: EXPRESSION

  • Prepare 96 deep well plate with 1ml of TB + antibiotic,
  • Inoculate well by taking 20ul of the overnight and place it in 1ml TB, do all inducible twice so to allow for induced verse uninducible expression
  • Grow on shaker at 37C for 1 hour
  • Induce all inducible constructs with 50ml of 10mM IPTG
  • Allow to grow for 18 hour at room temperature on a shaker

Day 3: DATA

  • Take overnights plates and spin on plate centrifuge for 20 minutes at 4000rpm
  • Pour of broth and resuspend in 1ml PBS 7.5 pH on plate shaker
  • Spin down PBS suspension for 20 minutes at 4000rpm
  • Pour of supernatant
  • Resuspend in 1ml PBS 7.5 pH on plate shaker
  • Take 100ul of PBS suspension and place into clear bottom plate reader plate
  • Take 100ul of PBS suspension and place into black plate reader plate
  • Read plates at 525nm and record data


← Back to Lab Protocols      

Retrieved from "http://2010.igem.org/Team:Washington/Protocols/VectorAssay"