|
Analytical Methods
To determine certain properties of proteins or contructed DNA fragments such as BioBricks, we have used different analytical methods. All used methods are listed below.
Agarose Gel Electrophoresis
The Agarose Gel Electrophoresis is used for separation of DNA or RNA fragments (e.g. after a PCR).
- take 5 µl of the PCR product
- mix with 1 µl loading dye
- apply onto agarose gel together with a marker
- run at 100 V for 35 minutes for a full gel
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The SDS-PAGE is used to determine certain features of the cells' proteome such as the strength of expression of a desired protein.
Cell Preparation
- lysis of cell pellet in lysis buffer
- centrifuge for 15 min at 13.000 rpm
- mix the supernatant with 2x lammli buffer with β-mercaptoethanol
- denatured for 5 min at 95°C
- sample to the gel
For some SDS-PAGEs, we used BioRad ready made gels.
Self-made SDS gels were made as described below:
1.5x Buffer
- 1.5 M Tris-Cl pH = 8.8
- in 1 L is 40 ml 10% SDS
Gels
|
| 0.75 mm 12% RUNNING Gel
| 1 mm 4% STACKING Gel
|
|
| 1x | 2x | 4x
| 1x | 2x | 4x
|
| H2O
| 1.65 ml | 3.3 ml | 6.6 ml
| 1.5 ml | 3 ml | 6 ml
|
| 1.5x Gel Buffer
| 1.3 ml | 2.6 ml | 5.2 ml
| 0.65 ml | 1.3 ml | 2.6 ml
|
| 30% Acrylamide (37.5:1)
| 2 ml | 4 ml | 8 ml
| 0.325 ml | 0.65 ml | 1.3 ml
|
| 10% APS
| 50 µl | 100 µl | 200 µl
| 25 µl | 50 µl | 100 µl
|
| TEMED
| 10 µl | 20 µl | 40 µl
| 5 µl | 10 µl | 20 µl
|
Run Gel
- apply the prepared samples together with a protein marker on the gel
- run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V
Bradford Assay
This assay is used for the determination of the protein concentration in a sample.
- mix the Bradford solution with ddH2O in a ratio of 1:4
- prepare about 10 solutions 1 ml, each between 125–1,000 μg/ml BSA for a standard curve
- use pure Bradford solution as a blank
- mix equal amounts of BSA and samples with unknown concentrations (1-3 µl) with 1 ml of 1x Bradford solution, vortex and incubate for 5 min. at room temperature
- measure the OD with a spectrophotometer at 595 nm
- build a standard curve within the linear range of the BSA data (concentration against OD)
- derive the concentration of your samples from the calibration curve
Measurement of Fluorescence
The measurement of fluorescence was performed using the Synergy Mx Microplate Reader (BioTek) and the corresponding Gen5 2.01 software. Unless stated otherwise following parameters were used:
- volume of sample in each well: 100 µl
- measurement of GFP/eGFP/sfGFP
- excitation wavelength: 496 ± 9 nm
- emission wavelength: 516 ± 9 nm
- measurement of iLOV
- excitation wavelength: 450 ± 9 nm
- emission wavelength: 495 ± 9 nm
The fluorescence is given out in arbitrary units (a.u.).
Measurement of Optical Density
Depending on the number of samples, two different devices were used for measurement of OD, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx Microplate Reader (BioTek) with the corresponding Gen5 2.01 software.
Unless stated otherwise following parameters were used for the plate reader measurements:
- volume of sample in each well: 100 µl
- OD600 with pathlength correction (including the height of liquid into the calculation of the optical density)
The respective culture medium was used as reference.
|
"
