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| <div class="left-text"> | | <div class="left-text"> |
| <h4>OD 600 Reference point</h4> | | <h4>OD 600 Reference point</h4> |
− | <p>❏ Add 100μl LUDOX into wells A1, B1, C1, D1 <br>❏ Add 100μl of dd H2O into wells A2, B2, C2, D2 <br>❏ Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements <br>❏ Record the data in the table below or in your notebook <br>❏ Import data into Excel sheet provided (OD600 reference point tab)<br><br> </p> | + | <p>❏ Add 100μL LUDOX into wells A1, B1, C1, D1 <br>❏ Add 100μL of dd H<sub>2</sub>O into wells A2, B2, C2, D2 <br>❏ Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements <br>❏ Record the data in the table below or in your notebook <br>❏ Import data into Excel sheet provided (OD600 reference point tab)<br><br> </p> |
| <h4>Particle Standard Curve</h4> | | <h4>Particle Standard Curve</h4> |
− | <p>❏ Obtain the tube labeled ”Silica Beads” from the InterLab test kit and vortex vigorously for 30 seconds. <br>❏ Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube <br>❏ Add 904μL of ddH2O to the microspheres <br>❏ Vortex well. This is your Microsphere Stock Solution. <br>❏ Repeat dilution series for rows B, C, D <br>❏ Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader<br>❏ Measure Abs600 of all samples in instrument <br>❏ Record the data in your notebook <br>❏ Import data into Excel sheet provided (particle standard curve tab) </p> | + | <p>❏ Obtain the tube labeled ”Silica Beads” from the InterLab test kit and vortex vigorously for 30 seconds. <br>❏ Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube <br>❏ Add 904μL of ddH<sub>2</sub>O to the microspheres <br>❏ Vortex well. This is your Microsphere Stock Solution. <br>❏ Repeat dilution series for rows B, C, D <br>❏ Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader<br>❏ Measure Abs600 of all samples in instrument <br>❏ Record the data in your notebook <br>❏ Import data into Excel sheet provided (particle standard curve tab) </p> |
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| <h4>Fluorescence standard curve</h4> | | <h4>Fluorescence standard curve</h4> |
− | <p>❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. <br>❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.<br>❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS<br>❏ Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 <br>❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1 <br>❏ Transfer 100 μl of fluorescein stock solution from A1 into A2. <br>❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3… <br>❏ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4... <br>❏ Mix A4 by pipetting up and down 3x and transfer 100 μl into A5... <br>❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6... <br>❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7... <br>❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8... <br>❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9... <br>❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10... <br>❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11... <br>❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste<br>❏ Repeat dilution series for rows B, C, D <br>❏ Measure fluorescence of all samples in instrument <br>❏ Record the data in your notebook <br>❏ Import data into Excel sheet provided (fluorescein standard curve tab) </p> | + | <p>❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. <br>❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.<br>❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS<br>❏ Add 100 μL of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 <br>❏ Add 200 μL of fluorescein 1x stock solution into A1, B1, C1, D1 <br>❏ Transfer 100 μL of fluorescein stock solution from A1 into A2. <br>❏ Mix A2 by pipetting up and down 3x and transfer 100 μL into A3… <br>❏ Mix A3 by pipetting up and down 3x and transfer 100 μL into A4... <br>❏ Mix A4 by pipetting up and down 3x and transfer 100 μL into A5... <br>❏ Mix A5 by pipetting up and down 3x and transfer 100 μL into A6... <br>❏ Mix A6 by pipetting up and down 3x and transfer 100 μL into A7... <br>❏ Mix A7 by pipetting up and down 3x and transfer 100 μL into A8... <br>❏ Mix A8 by pipetting up and down 3x and transfer 100 μL into A9... <br>❏ Mix A9 by pipetting up and down 3x and transfer 100 μL into A10... <br>❏ Mix A10 by pipetting up and down 3x and transfer 100 μL into A11... <br>❏ Mix A11 by pipetting up and down 3x and transfer 100 μL into liquid waste<br>❏ Repeat dilution series for rows B, C, D <br>❏ Measure fluorescence of all samples in instrument <br>❏ Record the data in your notebook <br>❏ Import data into Excel sheet provided (fluorescein standard curve tab) </p> |
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| <h4>Competent cells and Transformation</h4> | | <h4>Competent cells and Transformation</h4> |
− | <p>❏ Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. <br>❏ Thaw competent cells on ice<br>❏ Pipette 50µl of competent cells into 1.5ml tube<br>❏ Pipette 1µl of resuspended DNA into 1.5ml tube<br>❏ Pipette 1µl of control DNA into 2ml tube<br>❏ Close 1.5ml tubes, incubate on ice for 30min<br>❏ Heat shock tubes at 42°C for 45 sec<br>❏ Incubate on ice for 5min<br>❏ Pipette 950µl SOC media to each transformation<br>❏ Incubate at 37°C for 1 hours, shaking at 200-300rpm<br>❏ Pipette 100µL of each transformation onto petri plates<br>❏ Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates<br>❏ Incubate transformations overnight (14-18hr) at 37°C<br>❏ Pick single colonies for PCR<br>❏ Count colonies for control transformation</p> | + | <p>❏ Resuspend DNA in selected wells in the Distribution Kit with 10µL dH<sub>2</sub>0. <br>❏ Thaw competent cells on ice<br>❏ Pipette 50µL of competent cells into 1.5mL tube<br>❏ Pipette 1µL of resuspended DNA into 1.5mL tube<br>❏ Pipette 1µL of control DNA into 2mL tube<br>❏ Close 1.5mL tubes, incubate on ice for 30min<br>❏ Heat shock tubes at 42°C for 45 sec<br>❏ Incubate on ice for 5min<br>❏ Pipette 950µL SOC media to each transformation<br>❏ Incubate at 37°C for 1 hours, shaking at 200-300rpm<br>❏ Pipette 100µL of each transformation onto petri plates<br>❏ Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates<br>❏ Incubate transformations overnight (14-18hr) at 37°C<br>❏ Pick single colonies for PCR<br>❏ Count colonies for control transformation</p> |
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| <div class="left-text"> | | <div class="left-text"> |
| <h4>Cell measurement</h4> | | <h4>Cell measurement</h4> |
− | <p>❏ Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm<br>❏ Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor) <br>❏ Measure Abs 600 of these 1:10 diluted cultures <br>❏ Record the data in your notebook <br>❏ Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light). <br>❏ Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice. <br>❏ Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours. <br>❏ Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice. <br>❏ At the end of sampling point you need to measure your samples (Abs 600 and fluorescence measurement), see the below for details. <br>❏ Record data in your notebook <br>❏ Import data into Excel sheet provided (fluorescence measurement tab) </p> | + | <p>❏ Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium +Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm<br>❏ Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor) <br>❏ Measure Abs 600 of these 1:10 diluted cultures <br>❏ Record the data in your notebook <br>❏ Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 mL LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light). <br>❏ Take 500 µL samples of the diluted cultures at 0 hours into 1.5 mL eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice. <br>❏ Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours. <br>❏ Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 mL eppendorf tubes. Place samples on ice. <br>❏ At the end of sampling point you need to measure your samples (Abs 600 and fluorescence measurement), see the below for details. <br>❏ Record data in your notebook <br>❏ Import data into Excel sheet provided (fluorescence measurement tab) </p> |
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| <div class="left-text"> | | <div class="left-text"> |
| <h4>CFU per 0.1 OD600 E. coli cultures</h4> | | <h4>CFU per 0.1 OD600 E. coli cultures</h4> |
− | <p>❏ culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours <br>❏ Dilute the overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture. Check the OD600 and make sure it is 0.1<br>❏ Aseptically spead plate 100 μL on LB + Cam plates for those Final Dilution Factor is 8 x 104 or 8 x 105 or 8 x 106<br>❏ Incubate at 37°C overnight and count colonies after 18-20 hours of growth<br>❏ Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate. </p> | + | <p>❏ culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours <br>❏ Dilute the overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture. Check the OD600 and make sure it is 0.1<br>❏ Aseptically spead plate 100 μL on LB + Cam plates for those Final Dilution Factor is 8 x 10<sup>4</sup> or 8 x 10<sup>5</sup> or 8 x 10<sup>6</sup><br>❏ Incubate at 37°C overnight and count colonies after 18-20 hours of growth<br>❏ Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate. </p> |
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| <div class="left-text"> | | <div class="left-text"> |
| <h4>HD Images</h4> | | <h4>HD Images</h4> |
− | <p>Competent cells (Escherichia coli strain DH5α) <br>1ml LUDOX CL-X (provided in kit) <br>300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres) <br>LB (Luria Bertani) media <br>Fluorescein (provided in kit) <br>10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team) <br>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) <br>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) <br>Incubator at 37°C <br>1.5 ml eppendorf tubes for sample storage <br>Ice bucket with ice <br>Micropipettes and tips <br>96 well plate, black with clear flat bottom preferred (provided by team)</p> | + | <p>Competent cells (Escherichia coli strain DH5α) <br>1mL LUDOX CL-X (provided in kit) <br>300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10<sup>8</sup> microspheres) <br>LB (Luria Bertani) media <br>Fluorescein (provided in kit) <br>10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team) <br>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH) <br>50 mL Falcon tube (or equivalent, preferably amber or covered in foil to block light) <br>Incubator at 37°C <br>1.5 mL eppendorf tubes for sample storage <br>Ice bucket with ice <br>Micropipettes and tips <br>96 well plate, black with clear flat bottom preferred (provided by team)</p> |
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