Team:Bielefeld-Germany/Labjournal/Analytics
From 2013.igem.org
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- | <div class=" | + | <div class="nadh_headline"> |
- | <a name=" | + | <a name="nadh"><span style="color:#ff6600; padding-left=22px">[NADH-Assay]</span></a> |
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+ | For measurement of intracellular NADH concentration. | ||
+ | 1) Inoculating an overnight culture (30 mL with 1 mL of pre-culture). | ||
+ | 2) Centrifugation (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells. | ||
+ | 3) Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer. | ||
+ | 4) Resuspend pellet in 1 mL PBS buffer. | ||
+ | 5) Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm). | ||
+ | 6) Centrifugation for 10 min at maximum speed. | ||
+ | 7) Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader. | ||
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Revision as of 11:02, 26 September 2013
Analytics
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Pouring the polyacrylamide gel
- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED
- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel
- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel
- Layer isopropanol on top of the ge
- Leave the separating gel at room temperature for >60 minutes to polymerize
- Remove isopropanol and wait until the surface is dry
- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form
- Insert comb without getting bubbles stuck underneath
- Leave the gel at room temperature for >60 minutes to polymerize
For storage:
-Remove sealing and store the gel wrapped in moistened paper towel at 4°C
Preparing the sample
- Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)
- Heat for 5 minutes at 95 °C
Running the gel
- Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber
- Remove comb without destroying the gel pocket
- Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample
- Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel
- Raise amperage up to 20 mA for running the separating gel
- When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply