Revision as of 03:18, 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).

take 5 µl of the PCR product
mix with 1 µl loading dye
apply onto agarose gel together with a marker
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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 This assay is used for the determination of the protein concentration in a sample.
 * mix the Bradford solution with ddH{{sub|2}}O in a ratio of 1:4
-* prepare about 10 solutions 1&nbsp;mL, each between 125–1,000&nbsp;μg/mL BSA for a standard curve
+* prepare about 10 solutions 1&nbsp;ml, each between 125–1,000&nbsp;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&nbsp;µL) with 1&nbsp;mL of 1x&nbsp;Bradford solution, vortex and incubate for 5&nbsp;min. at room temperature
+* mix equal amounts of BSA and samples with unknown concentrations (1-3&nbsp;µl) with 1&nbsp;ml of 1x&nbsp;Bradford solution, vortex and incubate for 5&nbsp;min. at room temperature
 * measure the OD with a spectrophotometer at 595&nbsp;nm
 * build a standard curve within the linear range of the BSA data (concentration against OD)

Team:Aachen/Notebook/Protocols/Analytical methods

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