|
Analytical Methods
Agarose Gel Electrophoresis
Separation of DNA or RNA
- take 5µl of the PCR product
- mix with 1µl loading dye
- apply onto agarose gel together with a marker
- run at 120 mA for 40 minutes for a full gel
SDS-PAGE
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.
The recipe of the self-made SDS is as follows:
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 (BioTek) microplate reader and the Gen5 software.
- volume of sample in each well: 100µl
- measure GFP fluorescence at an excitation wavelength of 496 ± 9 nm and an emission wavelength at 516 ± 9 nm
Measurement of Optical Density
Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.
|
"
