Determination of dextran content of particles

1. Prepare solution of 80% phenol in water (Phenol stock is 89%)

2. Calculate how many wells will be used for the assay and based on this how much sulfuric acid and phenol is necessary, given 4μL of phenol and 200μL of sulfuric acid per well.

3. In a 50mL conical, prepare a stock solution of phenol and sulfuric acid based on calculated necessary volume in the correct ratio of 4 μL phenol : 200 μL sulfuric acid.

4. These procedures will change from experiment to experiment based on specific needs. Adjust amounts accordingly for desired sensitivity or data points.

Prepare a stock solution of dextran of 100mg/mL. Pipette 60 μL of this into one well of a 96-well plate. From there, perform two-fold serial dilutions down to a concentration of 0.39, making sure to discard 30 μL from the last well (so that each well contains 30 μL of solution). Pipette 30 μL of water into the next well as a control. This serial dilution will be for the standard curve. Make sure to pipette up and down each well in order to mix.

5. Pipette 30 μL of each test solution into a well in duplicate.

6. Add the stock solution of phenol and sulfuric acid to each well, mixing with the pipette each time.

7. Let stand for 10 min at room temperature, then place in 30°C incubator for 10 minutes.

8. Read on TECAN plate reader at 490nm with 10 sec shake. It may also be necessary to do a scan from 400-500 nm.

Created: 06/21/2007 Brian Lee