Team:Aachen/Notebook/Protocols/Analytical methods

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(→Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE))
 
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= Analytical Methods =
= 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==
== Agarose Gel Electrophoresis==
-
Separation of DNA or RNA
+
The Agarose Gel Electrophoresis is used for separation of DNA or RNA fragments (e.g. after a PCR).
-
# take 5µl of the PCR product
+
# take 5 µl of the PCR product
-
# mix with 1µl loading dye
+
# mix with 1 µl loading dye
# apply onto agarose gel together with a marker
# apply onto agarose gel together with a marker
-
# run at 120 mA for 40 minutes for a full gel
+
# 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.
-
== SDS-PAGE ==
+
'''Cell Preparation'''
-
=== Cell Preparation ===
+
* lysis of cell pellet in lysis buffer
* lysis of cell pellet in lysis buffer
* centrifuge for 15 min at 13.000 rpm
* centrifuge for 15 min at 13.000 rpm
* mix the supernatant with 2x lammli buffer with β-mercaptoethanol
* mix the supernatant with 2x lammli buffer with β-mercaptoethanol
-
* denatured for 5 min at 95 °C
+
* denatured for 5 min at 95°C
* sample to the gel  
* sample to the gel  
For some SDS-PAGEs, we used BioRad ready made gels.
For some SDS-PAGEs, we used BioRad ready made gels.
-
The recipe of the self-made SDS is as follows:
+
Self-made SDS gels were made as described below:
-
 
+
'''1.5x Buffer'''
-
=== 1.5x Buffer ===
+
* 1.5 M Tris-Cl pH = 8.8
* 1.5 M Tris-Cl pH = 8.8
-
* in 1 L is 40 ml 10 % SDS
+
* in 1 L is 40 ml 10% SDS
-
 
+
-
=== Gels ===
+
'''Gels'''
<center>
<center>
{| class="wikitable" style="text-align: right;"
{| class="wikitable" style="text-align: right;"
!  
!  
-
!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12 % RUNNING Gel  
+
!! style="border-left: 2px solid #404040;" colspan="3"|0.75&nbsp;mm 12% RUNNING Gel  
-
!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;mm 4 % STACKING Gel
+
!! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1&nbsp;mm 4% STACKING Gel
|-
|-
|  
|  
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|-
|-
| '''H{{sub|2}}O'''  
| '''H{{sub|2}}O'''  
-
| style="border-left: 2px solid #404040;"| 1.65&nbsp;mL || 3.3&nbsp;mL || 6.6&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 1.65&nbsp;ml || 3.3&nbsp;ml || 6.6&nbsp;ml
-
| style="border-left: 2px solid #404040;"| 1.5&nbsp;mL || 3&nbsp;mL || 6&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 1.5&nbsp;ml || 3&nbsp;ml || 6&nbsp;ml
|-
|-
| '''1.5x Gel Buffer'''  
| '''1.5x Gel Buffer'''  
-
| style="border-left: 2px solid #404040;"| 1.3&nbsp;mL || 2.6&nbsp;mL || 5.2&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 1.3&nbsp;ml || 2.6&nbsp;ml || 5.2&nbsp;ml
-
| style="border-left: 2px solid #404040;"| 0.65&nbsp;mL || 1.3&nbsp;mL || 2.6&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 0.65&nbsp;ml || 1.3&nbsp;ml || 2.6&nbsp;ml
|-
|-
-
| '''30 % Acrylamide (37.5:1)'''  
+
| '''30% Acrylamide (37.5:1)'''  
-
| style="border-left: 2px solid #404040;"| 2&nbsp;mL || 4&nbsp;mL || 8&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 2&nbsp;ml || 4&nbsp;ml || 8&nbsp;ml
-
| style="border-left: 2px solid #404040;"| 0.325&nbsp;mL || 0.65&nbsp;mL || 1.3&nbsp;mL
+
| style="border-left: 2px solid #404040;"| 0.325&nbsp;ml || 0.65&nbsp;ml || 1.3&nbsp;ml
|-
|-
-
| '''10 % APS'''  
+
| '''10% APS'''  
-
| style="border-left: 2px solid #404040;"| 50&nbsp;µL || 100&nbsp;µL || 200&nbsp;µL
+
| style="border-left: 2px solid #404040;"| 50&nbsp;µl || 100&nbsp;µl || 200&nbsp;µl
-
| style="border-left: 2px solid #404040;"| 25&nbsp;µL || 50&nbsp;µL || 100&nbsp;µL
+
| style="border-left: 2px solid #404040;"| 25&nbsp;µl || 50&nbsp;µl || 100&nbsp;µl
|-
|-
| '''TEMED'''  
| '''TEMED'''  
-
| style="border-left: 2px solid #404040;"| 10&nbsp;µL || 20&nbsp;µL || 40&nbsp;µL
+
| style="border-left: 2px solid #404040;"| 10&nbsp;µl || 20&nbsp;µl || 40&nbsp;µl
-
| style="border-left: 2px solid #404040;"| 5&nbsp;µL || 10&nbsp;µL || 20&nbsp;µL
+
| style="border-left: 2px solid #404040;"| 5&nbsp;µl || 10&nbsp;µl || 20&nbsp;µl
|-
|-
|}
|}
</center>
</center>
-
 
+
'''Run Gel'''
-
===Run Gel===
+
* apply the prepared samples together with a protein marker on the gel
* apply the prepared samples together with a protein marker on the gel
* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V
* run the gel for 10&nbsp;min at 60&nbsp;V and after that for ca. 60&nbsp;min at 120&nbsp;V
== Bradford Assay ==
== Bradford Assay ==
-
 
This assay is used for the determination of the protein concentration in a sample.  
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
* 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
* 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
* 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)  
* build a standard curve within the linear range of the BSA data (concentration against OD)  
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== Measurement of Fluorescence ==
== Measurement of Fluorescence ==
 +
The measurement of fluorescence was performed using the Synergy Mx Microplate Reader (BioTek) and the corresponding Gen5&nbsp;2.01 software. Unless stated otherwise following parameters were used:
 +
* volume of sample in each well: 100&nbsp;µl
 +
* measurement of GFP/eGFP/sfGFP
 +
** excitation wavelength: 496&nbsp;±&nbsp;9&nbsp;nm
 +
** emission wavelength: 516&nbsp;±&nbsp;9&nbsp;nm
 +
* measurement of iLOV
 +
** excitation wavelength: 450&nbsp;±&nbsp;9&nbsp;nm
 +
** emission wavelength: 495&nbsp;±&nbsp;9&nbsp;nm
 +
The fluorescence is given out in arbitrary units (a.u.).
-
The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
+
== 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&nbsp;2.01 software.
-
* volume of sample in each well: 100µl
+
Unless stated otherwise following parameters were used for the plate reader measurements:
-
* measure GFP fluorescence at an excitation wavelength of 496&nbsp;±&nbsp;9&nbsp;nm and an emission wavelength at 516&nbsp;±&nbsp;9&nbsp;nm
+
* volume of sample in each well: 100&nbsp;µl
-
 
+
* OD<sub>600</sub> with ''pathlength correction'' (including the height of liquid into the calculation of the optical density)
-
== 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.
+
The respective culture medium was used as reference.

Latest revision as of 03:42, 18 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 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.


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