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 | + | <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">Competent cells of E.coli DH5α were prepared following the provided protocol. Tested the OD of the cells using standard settings (see iGEM 2016 file),aiiming for a reading of 0.4-0.5. The first reading produced an average of 0.2, after 15 minutes the average increased to 2.5. The cells were spun down and re-suspended in TF-1 and TF-2 buffer. 100µl was aliquoted into 1 ml Eppendorf tubes and immediately dropped into liquid nitrogen. The cells were then left in the -80°C freezer. Chloramphenicol was made as a stock of 25mg/ml (weight measured was 76.6mg) and added to LB media to make plates for the iGEM interlab parts transformed with different constructs.</p> | <p id="pp">Competent cells of E.coli DH5α were prepared following the provided protocol. Tested the OD of the cells using standard settings (see iGEM 2016 file),aiiming for a reading of 0.4-0.5. The first reading produced an average of 0.2, after 15 minutes the average increased to 2.5. The cells were spun down and re-suspended in TF-1 and TF-2 buffer. 100µl was aliquoted into 1 ml Eppendorf tubes and immediately dropped into liquid nitrogen. The cells were then left in the -80°C freezer. Chloramphenicol was made as a stock of 25mg/ml (weight measured was 76.6mg) and added to LB media to make plates for the iGEM interlab parts transformed with different constructs.</p> | ||
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Measurements were then taken each hour for 6 hours by pipetting 100µl of each culture into the 96 well plate according to the iGEM layout for Abs600 and Fluorescence measurement and using the calibrated Tecan spectrometer.</p> | Measurements were then taken each hour for 6 hours by pipetting 100µl of each culture into the 96 well plate according to the iGEM layout for Abs600 and Fluorescence measurement and using the calibrated Tecan spectrometer.</p> | ||
| − | <p id="pp"> The protocol was improved by using a latin rectangle arrangement in another 96 well plate. A lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The | + | <p id="pp"> The protocol was improved by using a latin rectangle arrangement in another 96 well plate. A lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The Latin rectangle is designed so that no sample gets pipetted more than once in any row or column. The order in which the samples get arranged was worked out using the random number function of Microsoft Excel. Sterile LB was loaded into peripheral wells to lessen edge effects which can skew results due to high evaporation at the periphery. These adaptations aim to provide more accurate data.</p> |
| − | <p id="pp">The | + | <p id="pp">The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini vibrating incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator for 1.46 mins to warm up.</p> |
Revision as of 10:38, 19 October 2016
