IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Method) |
(→Method) |
||
Line 1: | Line 1: | ||
=Method= | =Method= | ||
*OD600 recorded in 1ml cuvette and converted into cell number using [[IGEM:Cambridge/2008/Notebook/Voltage/OD600_Calibration |calibration graph]] | *Five different mutant strains were selected for investigation, based on their growth data and theoretical prediction about potassium sequestration capability. [[IGEM:Cambridge/2008/Voltage/Mutant Strains |(See mutants information)]] | ||
*Six different concentrations of potassium were prepared by adding 1ml of 10x KCl stock solution to 9ml NOK broth, to make final [K+] of 0, 1, 5, 10, 50 & 200mM. | |||
*Each mutant was innoculated into 10ml NOK samples of different potassium concentrations, and grown overnight at 37°C in a shaking incubator. | |||
*In the morning, OD600 recorded in 1ml cuvette and converted into cell number using [[IGEM:Cambridge/2008/Notebook/Voltage/OD600_Calibration |calibration graph.]] Cells were kept on ice at all times after this. | |||
*500ul of this culture was extracted & centrifuged (5 min, low speed.) | *500ul of this culture was extracted & centrifuged (5 min, low speed.) | ||
*Supernatant removed and discarded (no further analysis due to viscosity problems) | *Supernatant removed and discarded (no further analysis due to viscosity problems) | ||
Line 6: | Line 9: | ||
*Pellet resuspended in 1.5ml SDW, cells then fractured by 3 rounds of freeze-thaw lysis in liquid nitrogen. | *Pellet resuspended in 1.5ml SDW, cells then fractured by 3 rounds of freeze-thaw lysis in liquid nitrogen. | ||
*Flame photometry performed on most sensitive setting [[IGEM:Cambridge/2008/Notebook/Voltage/Flame_Photometer_Calibration |(See calibration graph)]] | *Flame photometry performed on most sensitive setting [[IGEM:Cambridge/2008/Notebook/Voltage/Flame_Photometer_Calibration |(See calibration graph)]] | ||
*This was converted into data below using our 'Potassay'(potassium assay) spreadsheet. | |||
*Four repeats were performed during the course of the day. | |||
=Results= | =Results= |
Revision as of 07:45, 3 September 2008
Method
- Five different mutant strains were selected for investigation, based on their growth data and theoretical prediction about potassium sequestration capability. (See mutants information)
- Six different concentrations of potassium were prepared by adding 1ml of 10x KCl stock solution to 9ml NOK broth, to make final [K+] of 0, 1, 5, 10, 50 & 200mM.
- Each mutant was innoculated into 10ml NOK samples of different potassium concentrations, and grown overnight at 37°C in a shaking incubator.
- In the morning, OD600 recorded in 1ml cuvette and converted into cell number using calibration graph. Cells were kept on ice at all times after this.
- 500ul of this culture was extracted & centrifuged (5 min, low speed.)
- Supernatant removed and discarded (no further analysis due to viscosity problems)
- Pellet washed (resuspend in 1ml SDW and centrifuge 5 min, low speed.)
- Pellet resuspended in 1.5ml SDW, cells then fractured by 3 rounds of freeze-thaw lysis in liquid nitrogen.
- Flame photometry performed on most sensitive setting (See calibration graph)
- This was converted into data below using our 'Potassay'(potassium assay) spreadsheet.
- Four repeats were performed during the course of the day.