Difference between revisions of "Team:Florida Atlantic/InterLab"

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<h5>Transformation Procedure</h5>
 
<h5>Transformation Procedure</h5>
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add 5uL DNA
+
◻ added 5uL DNA
 
<br/>
 
<br/>
−
let sit 30 min on ice
+
◻ let sit 30 min on ice
 
<br/>
 
<br/>
−
heat shock at 42C for 30 sec
+
◻ heat shocked at 42C for 30 sec
 
<br/>
 
<br/>
−
place back on ice for 2 min
+
◻ placed back on ice for 2 min
 
<br/>
 
<br/>
−
add 450uL Luria Broth and incubate at 37c for 2 hours
+
◻ added 450uL Luria Broth and incubated at 37c for 2 hours
 
<br/>
 
<br/>
−
plate 100uL on LB/chloramphenicol plates
+
◻ plated 100uL on LB/chloramphenicol plates
 
<br/>
 
<br/>
−
grow overnight at 37C
+
◻ grew overnight at 37C
 
<br/>
 
<br/>
−
this was done for all 8 machines
+
◻ this was done for all 8 machines
 
<br/>
 
<br/>
  
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  Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
 
  Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
 
<br/>
 
<br/>
−
Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
+
Grew the cells overnight (16-18 hours) at 37°C and 220 rpm.
 
<br/>
 
<br/>
 
<h6>Day 3​:</h6>
 
<h6>Day 3​:</h6>
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◻ Imported data into Excel (Dilution Calculation​) Sheet_1 provided
 
◻ Imported data into Excel (Dilution Calculation​) Sheet_1 provided
 
<br/>
 
<br/>
−
◻ Dilute the cultures to a target OD600 of 0.02 (see the volume of preloading culture and
+
◻ 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 m​l LB medium + Chloramphenicol
 
media in Excel (Dilution Calculation​) Sheet_1) in 15 m​l 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/>
 
<br/>
−
◻ Incubate the cultures at 37°C and 220 rpm.
+
◻ Incubated the cultures at 37°C and 220 rpm.
 
<br/>
 
<br/>
−
◻ Take 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time
+
◻ Took 1 mL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time
−
point, you will take a sample from each of the 8 devices, two colonies per device, for a
+
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/>
−
◻ Place samples on ice.
+
◻ Placed samples on ice.
 
<br/>
 
<br/>
−
◻ At the end of sampling point you need to measure your samples (OD and Fl
+
◻ Measured samples (OD and Fl
−
measurement), see the below for details.
+
measurement)
 
<br/>
 
<br/>
−
◻ Record data in your notebook
+
◻ Recorded data in notebook
 
<br/>
 
<br/>
  

Revision as of 22:05, 29 September 2017

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Florida_Atlantic

Interlab Report


Member Participations

Calibrations:
- Completed by Douglas + Valentina on September 21st
Transformations:
- Completed by Douglas + Ariania and supervised by Dr. Pavlovic on September 26th
Cell Measurement:
- Completed by Douglas + Rachel S. and assisted by Eric and Daniela (Esiobu lab members) on September 27th, 28th, 29th

Methodology

Materials
Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/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, 2 different plates used: clear with flat bottom for absorbance; black with flat bottom for fluorescence
Devices (from InterLab Measurement Kit):
● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015

Calibration Procedure

Transformation Procedure
◻ added 5uL DNA
◻ let sit 30 min on ice
◻ heat shocked at 42C for 30 sec
◻ placed back on ice for 2 min
◻ added 450uL Luria Broth and incubated at 37c for 2 hours
◻ plated 100uL on LB/chloramphenicol plates
◻ grew overnight at 37C
◻ this was done for all 8 machines
Cell Measurement Procedure
Day 1​:
transform Escherichia coli DH5α with these following plasmids:
● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015
Day 2​:
Picked 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
Grew the cells overnight (16-18 hours) at 37°C and 220 rpm.
Day 3​:
Cell growth, sampling, and assay
◻ Set instrument to read OD600 (as OD calibration setting)
◻ Measured OD600 of the overnight cultures
◻ Recorded data
◻ Imported data into Excel (Dilution Calculation​) Sheet_1 provided
◻ 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 m​l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
◻ Incubated the cultures at 37°C and 220 rpm.
◻ 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)
◻ Placed samples on ice.
◻ Measured samples (OD and Fl measurement)
◻ Recorded data in notebook