Team:Aachen/Notebook/Protocols/Analytical methods

From 2014.igem.org

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Revision as of 03:16, 17 October 2014

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).

  1. take 5 µl of the PCR product
  2. mix with 1 µl loading dye
  3. apply onto agarose gel together with a marker
  4. run at 120 mA for 40 minutes for a full gel

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The SDS-PAGE was used to determine certain features of the cells' proteom 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 (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.


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