Difference between revisions of "Team:Exeter/Interlab"
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<p id="pp">InterLab study began by measuring the standard LUDOX Abs600. Pipetted LUDOX and water into two separate columns of a 96 well plate and measured the absorbance of the 4 replicates at 600 nm on the standard Tecan mode.</p> | <p id="pp">InterLab study began by measuring the standard LUDOX Abs600. Pipetted LUDOX and water into two separate columns of a 96 well plate and measured the absorbance of the 4 replicates at 600 nm on the standard Tecan mode.</p> | ||
| − | <p id="pp">A serial dilution of FITC provided in the interkab kit was prepared using PBS. Fluorescence of all samples was measured in standard measurement modes. Measurements were repeated to produce a series of standard curves. The fluorescence readings were taken at 26.4°C at 477 nm to 515 nm excitation and emission respectively. Gain setting measured at 46, 56, 66. A setting change of 10 units makes a significant difference to the fluorescence data. The plate reader was run at optimum gain setting. This set gain to 76, which was too high for these measurements as the weakest dilution had a lower fluorescence than the pure PBS. Ran the reader at 37°C and 56 gain to produce our optimal standard curve.</p> | + | <p id="pp">A serial dilution of FITC provided in the interkab kit was prepared using PBS. Fluorescence of all samples was measured in standard measurement modes. Measurements were repeated to produce a series of standard curves. The fluorescence readings were taken at 26.4°C at 477 nm to 515 nm excitation and emission respectively. Gain setting measured at 46, 56, 66. A setting change of 10 units makes a significant difference to the fluorescence data (Fig. 1). The plate reader was run at optimum gain setting. This set gain to 76, which was too high for these measurements as the weakest dilution had a lower fluorescence than the pure PBS. Ran the reader at 37°C and 56 gain to produce our optimal standard curve (Fig. 2).</p> |
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<p id="pp">An initial OD600 reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator.</p> | <p id="pp">An initial OD600 reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator.</p> | ||
| − | <p id="pp">The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. | + | <p id="pp">The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. 10,000 events were acquired from calibration beads and from each biological sample.</p> |
<p id="pp">An initial OD reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator. </p> | <p id="pp">An initial OD reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator. </p> | ||
Revision as of 14:38, 19 October 2016
