Team:British Columbia/Protocols/ConsortiaFluor

From 2012.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 21: Line 21:
<div id="sponsormap"><img align="left" src="https://static.igem.org/mediawiki/2012/d/db/Ubcigemnotebookmenu2.jpg" usemap="#sponsormap" alt="UBC iGEM 2012 protocols"> </div><div id=protocol></html>
<div id="sponsormap"><img align="left" src="https://static.igem.org/mediawiki/2012/d/db/Ubcigemnotebookmenu2.jpg" usemap="#sponsormap" alt="UBC iGEM 2012 protocols"> </div><div id=protocol></html>
-
 
-
 
<h1>Consortia Fluorescence Monitoring</h1>
<h1>Consortia Fluorescence Monitoring</h1>
-
1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100mg/mL) .  
+
#Grow overnight 5mL cultures of auxotrophs(MetA<sup>-</sup>, TrpA<sup>-</sup>, and TyrA<sup>-</sup>)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .  
-
 
+
#Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.  
-
2. Spin down cells using a centrifuge (1600g, 10min). Pour out LB supernatant.  
+
#Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
-
 
+
#Perform step 3 three times.  
-
3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600g, 10min)
+
#Resuspend in 5 mL M9 Media.
-
 
+
#Measure the respective O.D 600 with a spectrometer.
-
4. Perform step 3 three times.  
+
#Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200 μL M9 with ampicillin (100 mg/mL).
-
 
+
#Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.
-
5. Resuspend in 5mL M9 Media.
+
-
 
+
-
6. Measure the respective O.D 600 with a spectrometer.
+
-
 
+
-
7. Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200uL M9 with ampicillin (100mg/mL).
+
-
 
+
-
8. Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.
+
-
 
+
-
 
+
-
 
+
-
 
+
-
==Excitation and Emission Wavelengths of Fluorescent Proteins==
+
<h1>Excitation and Emission Wavelengths of Fluorescent Proteins</h1>
-
EYFP - Excitation: 514nm    Emission: 527nm  
+
*EYFP - Excitation: 514nm    Emission: 527nm  
-
ECFP - Excitation: 439nm    Emission: 476nm
+
*ECFP - Excitation: 439nm    Emission: 476nm
-
ERFP - Excitation: 584nm    Emission: 607nm
+
*ERFP - Excitation: 584nm    Emission: 607nm

Latest revision as of 03:42, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

Consortia Fluorescence Monitoring

  1. Grow overnight 5mL cultures of auxotrophs(MetA-, TrpA-, and TyrA-)with their respective fluorescent proteins in LB media and ampicillin (100 mg/mL) .
  2. Spin down cells using a centrifuge (1600 g, 10 min). Pour out LB supernatant.
  3. Wash and resuspend in 5mL M9 media. Spin down cells using a centrifuge (1600 g, 10 min)
  4. Perform step 3 three times.
  5. Resuspend in 5 mL M9 Media.
  6. Measure the respective O.D 600 with a spectrometer.
  7. Inoculate appropriate co-culture of auxotrophs (each with a starting O.D.600 of 0.05) in a 96 well plate containing 200 μL M9 with ampicillin (100 mg/mL).
  8. Incubate in Tecan plate scanner, taking measurements of O.D.600 and fluorescence outputs for time intervals of every 15 minutes for 24 hours.

Excitation and Emission Wavelengths of Fluorescent Proteins

  • EYFP - Excitation: 514nm Emission: 527nm
  • ECFP - Excitation: 439nm Emission: 476nm
  • ERFP - Excitation: 584nm Emission: 607nm