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| === QuikChange === | | === QuikChange === |
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- | {{Team:Aachen/BlockSeparator}}
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- | = Analytical methods =
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- | == Agarose gel electrophoresis==
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- | Separation of DNA or RNA
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- | # take 5µl of the PCR product
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- | # mix with 1µl loading dye
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- | # apply onto agarose gel together with a marker
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- | # run at 120°C for 40 minutes for a full gel
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- | == SDS-PAGE ==
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- | === Cell preparation ===
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- | * lysis cell pellet in lysis buffer
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- | * centrifuge for 15 min at 13.000 rpm
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- | * mix the supernatant with 2x lammli buffer with β-mercaptoethanol
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- | * denatured for 5 min at 95 °C
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- | * sample to the gel
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- | For some SDS-PAGEs, we used BioRad ready made gels.
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- | The recipe of the self-made SDS is as follows:
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- | === 1.5x Buffer ===
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- | * 1.5 M Tris-Cl pH = 8.8
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- | * in 1 L is 40 ml 10 % SDS
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- | === Gels ===
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- | <center>
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- | {| class="wikitable" style="text-align: right;"
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- | !
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- | !! style="border-left: 2px solid #404040;" colspan="3"|0.75 mm 12 % RUNNING Gel
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- | !! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1 mm 4 % STACKING Gel
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- | |-
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- | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
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- | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
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- | | '''H{{sub|2}}O'''
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- | | style="border-left: 2px solid #404040;"| 1.65 mL || 3.3 mL || 6.6 mL
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- | | style="border-left: 2px solid #404040;"| 1.5 mL || 3 mL || 6 mL
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- | |-
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- | | '''1.5x Gel Buffer'''
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- | | style="border-left: 2px solid #404040;"| 1.3 mL || 2.6 mL || 5.2 mL
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- | | style="border-left: 2px solid #404040;"| 0.65 mL || 1.3 mL || 2.6 mL
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- | |-
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- | | '''30 % Acrylamide (37.5:1)'''
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- | | style="border-left: 2px solid #404040;"| 2 mL || 4 mL || 8 mL
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- | | style="border-left: 2px solid #404040;"| 0.325 mL || 0.65 mL || 1.3 mL
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- | | '''10 % APS'''
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- | | style="border-left: 2px solid #404040;"| 50 µL || 100 µL || 200 µL
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- | | style="border-left: 2px solid #404040;"| 25 µL || 50 µL || 100 µL
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- | |-
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- | | '''TEMED'''
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- | | style="border-left: 2px solid #404040;"| 10 µL || 20 µL || 40 µL
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- | | style="border-left: 2px solid #404040;"| 5 µL || 10 µL || 20 µL
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- | |-
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- | |}
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- | </center>
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- | ==Run gel==
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- | * apply the prepared samples together with a protein marker on the gel
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- | * run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V
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- | == Bradford assay ==
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- | Determination of protein concentration
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- | == Measurement of fluorescence ==
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- | The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
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- | * volume of sample in each well: 100µl
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- | * measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm
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- | == Measurement of optical density ==
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- | 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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