Difference between revisions of "Team:CCU Taiwan/InterLab"

(5 intermediate revisions by the same user not shown)
Line 239: Line 239:
  
 
     <p>
 
     <p>
      microplate reader FLUOstar Omega</br>
+
<ul class=”a”;padding-left:10px>
emission filter: 520 nm</br>
+
    <li>microplate reader FLUOstar Omega</li>
excitation filter: 485 nm
+
<li>emission filter: 520 nm</li>
 +
<li>excitation filter: 485 nm</li></ul>
 
     </p>
 
     </p>
  
Line 250: Line 251:
 
<h3>Material</h3>
 
<h3>Material</h3>
 
<p>
 
<p>
Fluorescein sodium salt</br>
+
<ul class=”a”;padding-left:10px>
1xPBS</br>
+
<li>Fluorescein sodium salt</li>
Tissue culture testplate (black with flat bottom)
+
<li>1xPBS</li>
 +
<li>Tissue culture testplate (black with flat bottom)</li></ul>
 
</p>
 
</p>
  
Line 262: Line 264:
 
 
 
<ol><li>Prepare fluorescein stock solution</li></ol>
 
<ol><li>Prepare fluorescein stock solution</li></ol>
<p>
+
<p><ul class=”a”;padding-left:10px>
1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</br>  
+
<li>1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li>  
2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </br>
+
<li>2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </li>
3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM </br>
+
<li>3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM </li>
 +
</ul>
 
</p>
 
</p>
 
<ol><li>Serial dilutions</li></ol>
 
<ol><li>Serial dilutions</li></ol>
<p>
+
<p><ul class=”a”;padding-left:10 px>
1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.</br>
+
<li>1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.</li>
2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.</br>
+
<li>2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.</li>
3. Transfer 100 μl of fluorescein stock solution from A1 into A2.</br>
+
<li>3. Transfer 100 μl of fluorescein stock solution from A1 into A2.</li>
4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </br>
+
<li>4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </li>
5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </br>
+
<li>5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </li>
6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </br>
+
<li>6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </li>
7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </br>
+
<li>7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </li>
8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </br>
+
<li>8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </li>
9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </br>
+
<li>9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </li>
10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </br>
+
<li>10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </li>
11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</br>  
+
<li>11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</li>  
12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </br>
+
<li>12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </li>
13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
+
<li>13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
   (Caution: Do not to continue serial dilution into column 12.)</br>
+
   (Caution: Do not to continue serial dilution into column 12.)</li>
 +
</ul>
 
</p>
 
</p>
<ol><li>repeat serial dilute for Row B、D、E</strong></li></ol>
+
<ol><li>Repeat serial dilute for Row B、D、E</strong></li></ol>
 
<ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol>
 
<ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol>
 
<ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol>
 
<ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol>
 +
 
 
 
<br/><br/>
 
<br/><br/>
Line 312: Line 317:
 
<h3>Plate reader</h3>
 
<h3>Plate reader</h3>
  
     <p>
+
     <p><ul class=”a”;padding-left:10px>
Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</br>
+
 
Filter: 595 nm</br>
+
<li>Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</li>
 +
<li>Filter: 595 nm</li>
 +
</ul>
 
     </p>
 
     </p>
  
Line 322: Line 329:
 
   <div class="aaa"></div>
 
   <div class="aaa"></div>
 
<h3>Material</h3>
 
<h3>Material</h3>
<p>
+
<p><ul class=”a”;padding-left:10px>
1 ml LUDOX</br>
+
<li>1 ml LUDOX</li>
mQH<sub>2</sub>O</br>
+
<li>mQH<sub>2</sub>O </li>
96 well cell culture plate (clear with flat-bottom)
+
<li>96 well cell culture plate (clear with flat-bottom)</li>
 
</p>
 
</p>
  
Line 335: Line 342:
  
 
<p>
 
<p>
1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</br>
+
<ul class="a">
2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</br>
+
<li>1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</li>
3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</br>
+
<li>2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</li>
4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</br>
+
<li>3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li>
 +
<li>4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</li>
 +
</ul>
 
</p>
 
</p>
 +
 
 
 
</div>
 
</div>
Line 361: Line 371:
 
<h3>Material</h3>
 
<h3>Material</h3>
 
<p>
 
<p>
Competent cells ( Escherichia coli strain DH5α)</br>
+
<ul class=”a”; padding-left:10px>
LB (Luria Bertani) media</br>
+
<li>Competent cells ( Escherichia coli strain DH5α)</li>
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</br>
+
<li>LB (Luria Bertani) media</li>
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</br>
+
<li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</li>
Incubator at 37°C</br>
+
<li>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block <li>light)</li>
1.5 ml eppendorf tubes for sample storage</br>
+
<li>Incubator at 37°C</li>
Ice bucket with ice</br>
+
<li>1.5 ml eppendorf tubes for sample storage</li>
Pipettes</br>
+
<li>Ice bucket with ice</li>
96 well plate(cell culture 96 well plate、tissue culture testplate)</br>
+
<li>Pipettes</li>
Devices (from InterLab Measurement Kit):</br>
+
<li>96 well plate(cell culture 96 well plate、tissue culture testplate)</li>
1. Negative control(BBa_R0040)</br>
+
<li>Devices (from InterLab Measurement Kit):</li>
2. Positive control(J23151+B0032+E0040+B0010+B0012)</br>
+
<li>&nbsp;&nbsp;1. Negative control(BBa_R0040)</li>
3. Test Device 1: J23101+I13504</br>
+
<li>&nbsp;&nbsp;2. Positive control(J23151+B0032+E0040+B0010+B0012)</li>
4. Test Device 2: J23106+I13504</br>
+
<li>&nbsp;&nbsp;3. Test Device 1: J23101+I13504</li>
5. Test Device 3: J23117+I13504</br>
+
<li>&nbsp;&nbsp;4. Test Device 2: J23106+I13504</li>
6. Test Device 4: J23101+BCD2+E0040+B0015</br>
+
<li>&nbsp;&nbsp;5. Test Device 3: J23117+I13504</li>
7. Test Device 5: J23106+BCD2+E0040+B0015</br>
+
<li>&nbsp;&nbsp;6. Test Device 4: J23101+BCD2+E0040+B0015</li>
8. Test Device 6: J23117+BCD2+E0040+B0015</br>
+
<li>&nbsp;&nbsp;7. Test Device 5: J23106+BCD2+E0040+B0015</li>
 +
<li>&nbsp;&nbsp;8. Test Device 6: J23117+BCD2+E0040+B0015</li>
 +
</ul>
 
</p>
 
</p>
  
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<p>
 
<p>
1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</br>
+
<ul class=”a”;padding-left:10 px>
&nbsp;(Transformation protocol is from iGEM)</br>
+
<li>1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</li>
2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</br>
+
<li>&nbsp;(Transformation protocol is from iGEM)</li>
3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</br>
+
<li>2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB <li>medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</li>
4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</br>
+
<li>3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</li>
5. Incubate the cultures at 37°C and 170 rpm.</br>
+
<li>4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</li>
6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</br>
+
<li>5. Incubate the cultures at 37°C and 170 rpm.</li>
7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</br>
+
<li>6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</li>
 +
<li>7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</li></ul>
 
</p>
 
</p>
  

Revision as of 04:43, 15 December 2017

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Fluorescein Fluorescence standard curve

Plate reader

  • microplate reader FLUOstar Omega
  • emission filter: 520 nm
  • excitation filter: 485 nm

Material

  • Fluorescein sodium salt
  • 1xPBS
  • Tissue culture testplate (black with flat bottom)

Method

  1. Prepare fluorescein stock solution

  • 1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
  • 2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
  • 3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM

  1. Serial dilutions

  • 1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.
  • 2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.
  • 3. Transfer 100 μl of fluorescein stock solution from A1 into A2.
  • 4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
  • 5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.
  • 6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.
  • 7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.
  • 8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.
  • 9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.
  • 10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.
  • 11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.
  • 12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
  • 13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste. (Caution: Do not to continue serial dilution into column 12.)

  1. Repeat serial dilute for Row B、D、E
  1. Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook
  1. Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided


Data result







OD600 Reference point

Plate reader

  • Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer
  • Filter: 595 nm

Material

  • 1 ml LUDOX
  • mQH2O
  • 96 well cell culture plate (clear with flat-bottom)

Method

  • 1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
  • 2. Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
  • 3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
  • 4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided

Data result



Cell measure

Material

  • Competent cells ( Escherichia coli strain DH5α)
  • LB (Luria Bertani) media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)
  • 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block
  • light)
  • Incubator at 37°C
  • 1.5 ml eppendorf tubes for sample storage
  • Ice bucket with ice
  • Pipettes
  • 96 well plate(cell culture 96 well plate、tissue culture testplate)
  • Devices (from InterLab Measurement Kit):
  •   1. Negative control(BBa_R0040)
  •   2. Positive control(J23151+B0032+E0040+B0010+B0012)
  •   3. Test Device 1: J23101+I13504
  •   4. Test Device 2: J23106+I13504
  •   5. Test Device 3: J23117+I13504
  •   6. Test Device 4: J23101+BCD2+E0040+B0015
  •   7. Test Device 5: J23106+BCD2+E0040+B0015
  •   8. Test Device 6: J23117+BCD2+E0040+B0015

Method

  • 1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.
  •  (Transformation protocol is from iGEM)
  • 2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB
  • medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.
  • 3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures
  • 4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).
  • 5. Incubate the cultures at 37°C and 170 rpm.
  • 6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice
  • 7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above

Data result