Team:Bielefeld-Germany/Labjournal/Analytics

From 2013.igem.org

(Difference between revisions)
m
m
Line 81: Line 81:
</div>
</div>
<div class="nadh" >
<div class="nadh" >
 +
<p> This method has been used for measurement of intracellular NADH concentration.
 +
<br><b><u>Protocol: </u></b>
 +
<p> • Inoculate an overnight culture (30 mL with 1 mL of pre-culture) </p>
 +
<p> • Centrifugate (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 </p>
 +
<p> • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer</p>
 +
<p> • Resuspend pellet in 1 mL PBS buffer</p>
 +
<p> • Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm) </p>
 +
<p> • Centrifugation for 10 min at maximum speed</p>
 +
<p> • Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader</p>
-
For measurement of intracellular NADH concentration.
+
<br><b><u>Tecan Infinite® M200 platereader parameters: </u></b>
-
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.
+
<p> • Sample volume = 100 μL clear supernatant </p>
-
3) Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer.
+
<p> • Excitation = 340 nm</p>
-
4) Resuspend pellet in 1 mL PBS buffer.
+
<p> • Emission = 460 nm</p>
-
5) Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm).
+
<p> • Concentration calculation by NADH calibration curve</p>
-
6) Centrifugation for 10 min at maximum speed.
+
-
7) Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader.
+

Revision as of 11:14, 26 September 2013







Analytics


[SDS-PAGE]

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



[NADH-Assay]

This method has been used for measurement of intracellular NADH concentration.
Protocol:

• Inoculate an overnight culture (30 mL with 1 mL of pre-culture)

• Centrifugate (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

• Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer

• Resuspend pellet in 1 mL PBS buffer

• Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)

• Centrifugation for 10 min at maximum speed

• Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader


Tecan Infinite® M200 platereader parameters:

• Sample volume = 100 μL clear supernatant

• Excitation = 340 nm

• Emission = 460 nm

• Concentration calculation by NADH calibration curve




Retrieved from "http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/Analytics"