Difference between revisions of "Team:Florida Atlantic/InterLab"
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| + | <img src="https://static.igem.org/mediawiki/2017/archive/2/20/20170929214604%21T--Florida_Atlantic--owlbanner.png"> | ||
<form><fieldset> | <form><fieldset> | ||
| − | <h1>Interlab Report</h1> | + | <h1 style="font-size: 26px">Interlab Report</h1> |
<br/> | <br/> | ||
| − | <center><h4>Member Participations</h4></center> | + | <center><h4 style="font-size: 22px">Member Participations</h4></center> |
| − | <h5>Calibrations:</h5> | + | <h5 style="font-size: 20px">Calibrations:</h5> |
| − | - Completed by Douglas + Valentina on September 21st | + | <p style="font-size: 18px"> - Completed by Douglas + Valentina on September 21st</p> |
<br/> | <br/> | ||
| − | <h5>Transformations:</h5> | + | <h5 style="font-size: 20px">Transformations:</h5> |
| − | - Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th | + | <p style="font-size: 18px"> - Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th</p> |
<br/> | <br/> | ||
| − | <h5>Cell Measurement:</h5> | + | <h5 style="font-size: 20px">Cell Measurement:</h5> |
| − | - Completed by Douglas + Rachel S. and assisted by | + | <p style="font-size: 18px"> - Completed by Douglas + Rachel S. and assisted by Erick and Daniela (Esiobu lab members) on September 27th, 28th, 29th</p> |
<br/> | <br/> | ||
<br/> | <br/> | ||
| − | <center><h4>Methodology</h4></center> | + | <center><h4 style="font-size: 22px">Methodology</h4></center> |
| − | <h5>Materials</h5> | + | <h5 style="font-size: 20px">Materials</h5> |
| − | Competent cells (Escherichia coli strain DH5α) | + | <p style="font-size: 18px"> ● Competent cells (Escherichia coli strain DH5α) |
<br/> | <br/> | ||
| − | LB (Luria Bertani) media | + | ● LB (Luria Bertani) media |
<br/> | <br/> | ||
| − | Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL) | + | ● Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL) |
<br/> | <br/> | ||
| − | 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) | + | ● 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) |
<br/> | <br/> | ||
| − | Incubator at 37°C | + | ● Incubator at 37°C |
<br/> | <br/> | ||
| − | 1.5 ml eppendorf tubes for sample storage | + | ● 1.5 ml eppendorf tubes for sample storage |
<br/> | <br/> | ||
| − | Ice bucket with ice | + | ● Ice bucket with ice |
<br/> | <br/> | ||
| − | Pipettes | + | ● Pipettes |
<br/> | <br/> | ||
| − | 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence | + | ● 96 well plate, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence </p> |
<br/> | <br/> | ||
| − | <h6>Devices (from InterLab Measurement Kit): </h6> | + | <h6 style="font-size: 20px">Devices (from InterLab Measurement Kit): </h6> |
| − | ● Positive control | + | <p style="font-size: 18px">● Positive control |
<br/> | <br/> | ||
● Negative control | ● Negative control | ||
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● Test Device 5: J23106.BCD2.E0040.B0015 | ● Test Device 5: J23106.BCD2.E0040.B0015 | ||
<br/> | <br/> | ||
| − | ● Test Device 6: J23117.BCD2.E0040.B0015 | + | ● Test Device 6: J23117.BCD2.E0040.B0015</p> |
<br/> | <br/> | ||
<br/> | <br/> | ||
| − | <h5>Calibration Procedure</h5> | + | <h5 style="font-size: 20px">Calibration Procedure</h5> |
| + | <p style="font-size: 18px">◻ Added 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette) | ||
| + | <br/> | ||
| + | ◻ Added 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette) | ||
| + | <br/> | ||
| + | ◻ Measured absorbance 600 nm of all samples in all standard measurement modes in | ||
| + | instrument | ||
| + | <br/> | ||
| + | ◻ Recorded the data in the table below or in notebook | ||
| + | <br/> | ||
| + | ◻ Imported data into Excel (OD600 reference point tab) Sheet_1 provided </p> | ||
<br/> | <br/> | ||
| − | <h5>Transformation Procedure</h5> | + | <h5 style="font-size: 20px">Transformation Procedure</h5> |
| − | + | <p style="font-size: 18px"> ◻ added 5uL DNA | |
<br/> | <br/> | ||
| − | let sit 30 min on ice | + | ◻ let sit 30 min on ice |
<br/> | <br/> | ||
| − | heat | + | ◻ heat shocked at 42C for 30 sec |
<br/> | <br/> | ||
| − | + | ◻ placed back on ice for 2 min | |
<br/> | <br/> | ||
| − | + | ◻ added 450uL Luria Broth and incubated at 37c for 2 hours | |
<br/> | <br/> | ||
| − | + | ◻ plated 100uL on LB/chloramphenicol plates | |
<br/> | <br/> | ||
| − | + | ◻ grew overnight at 37C | |
<br/> | <br/> | ||
| − | this was done for all 8 machines | + | ◻ this was done for all 8 machines</p> |
<br/> | <br/> | ||
| − | <h5>Cell Measurement Procedure</h5> | + | <h5 style="font-size: 20px">Cell Measurement Procedure</h5> |
| − | Day 1: transform Escherichia coli DH5α with these following plasmids: | + | </br> |
| − | ● Positive control | + | <h6 style="font-size: 19px">Day 1:</h6> |
| + | <p style="font-size: 18px">transform Escherichia coli DH5α with these following plasmids:</p> | ||
| + | <br/> | ||
| + | <p style="font-size: 18px">● Positive control | ||
| + | <br/> | ||
● Negative control | ● Negative control | ||
| + | <br/> | ||
● Test Device 1: J23101+I13504 | ● Test Device 1: J23101+I13504 | ||
| + | <br/> | ||
● Test Device 2: J23106+I13504 | ● Test Device 2: J23106+I13504 | ||
| + | <br/> | ||
● Test Device 3: J23117+I13504 | ● Test Device 3: J23117+I13504 | ||
| + | <br/> | ||
● Test Device 4: J23101.BCD2.E0040.B0015 | ● Test Device 4: J23101.BCD2.E0040.B0015 | ||
| + | <br/> | ||
● Test Device 5: J23106.BCD2.E0040.B0015 | ● Test Device 5: J23106.BCD2.E0040.B0015 | ||
| − | ● Test Device 6: J23117.BCD2.E0040.B0015 | + | <br/> |
| − | Day 2: Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. | + | ● Test Device 6: J23117.BCD2.E0040.B0015</p> |
| − | + | <br/> | |
| − | Day 3: Cell growth, sampling, and assay | + | <h6 style="font-size: 19px">Day 2:</h6> |
| + | <p style="font-size: 18px"> Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. | ||
| + | <br/> | ||
| + | Grew the cells overnight (16-18 hours) at 37°C and 220 rpm. | ||
| + | <br/> | ||
| + | <h6 style="font-size: 19px">Day 3:</h6> | ||
| + | <p style="font-size: 18px"> Cell growth, sampling, and assay | ||
| + | <br/> | ||
◻ Set instrument to read OD600 (as OD calibration setting) | ◻ Set instrument to read OD600 (as OD calibration setting) | ||
| + | <br/> | ||
◻ Measured OD600 of the overnight cultures | ◻ Measured OD600 of the overnight cultures | ||
| + | <br/> | ||
◻ Recorded data | ◻ Recorded data | ||
| + | <br/> | ||
◻ Imported data into Excel (Dilution Calculation) Sheet_1 provided | ◻ Imported data into Excel (Dilution Calculation) Sheet_1 provided | ||
| − | ◻ | + | <br/> |
| + | ◻ Diluted the cultures to a target OD600 of 0.02 (see the volume of preloading culture and | ||
media in Excel (Dilution Calculation) Sheet_1) in 15 ml LB medium + Chloramphenicol | media in Excel (Dilution Calculation) Sheet_1) in 15 ml LB medium + Chloramphenicol | ||
in 50 mL falcon tube (amber, or covered with foil to block light). | in 50 mL falcon tube (amber, or covered with foil to block light). | ||
| − | ◻ | + | <br/> |
| − | ◻ | + | ◻ Incubated the cultures at 37°C and 220 rpm. |
| − | point, | + | <br/> |
| + | ◻ Took 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time | ||
| + | point, sampled from each of the 8 devices, two colonies per device, for a | ||
total of 16 samples per time point) | total of 16 samples per time point) | ||
| − | |||
| − | |||
| − | |||
| − | |||
<br/> | <br/> | ||
| − | + | ◻ Placed samples on ice. | |
| − | + | <br/> | |
| − | + | ◻ Measured samples (OD and Fl | |
| + | measurement) | ||
| + | <br/> | ||
| + | ◻ Recorded data in notebook</p> | ||
| + | <br/> | ||
| + | <center><h4 style="font-size: 22px">Data</h4></center> | ||
| + | <img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/archive/d/d3/20171026205230%21T--Florida_Atlantic--interlabdata.png" width=200; height=700;> | ||
| + | <br/> | ||
| + | <center><h4 style="font-size: 22px">Results</h4></center> | ||
| + | <br/> | ||
| + | <center><h5>Fluorescence of GFP engineered E.coli Graphs</h5></center> | ||
| + | <div class="column half_size" > | ||
| + | <center><img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/d/dd/Interlab_Data_Graph.png" width=200; height=400;></center> | ||
| + | </div> | ||
| + | <div class="column half_size" > | ||
| + | <center><img style="max-width:95%;border:3px solid darkred;" src="https://static.igem.org/mediawiki/2017/c/c9/Interlab_Data_Graph2.png" width=200; height=400";></center> | ||
</div> | </div> | ||
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Latest revision as of 20:49, 1 November 2017
Florida_Atlantic
