User:Allison K. Alix/Notebook/CHEM-581/2013/04/03

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Objective

-Mix hydrogels with remaining indicators (bromocresol purple, phenolphthalein, cresol red)

-View hydrogels containing fluorescent dye (R6G) under fluorescent microscope

-Prepare new sets of hydrogels

Procedures

Part 1: Addition of indicators to hydrogel solutions

1) Add 50μL of phenolphthalein to 5mg hydrogel in 1.5mL buffer prepared on 03/29/2013

2) Add 25μL bromocresol purple to 5mg hydrogels in 1.5mL phosphate buffer

3) Add 75μL 0.02% cresol red to 5mg hydrogels in 1.5mL phosphate buffer

4) Allow time for hydrogels to absorb indicator

Part 2: Create concentration gradient for fluroescence calibration of microscope

1) Prepare a solution of 10μL FluoSpheres carboxylate modified microspheres, (0.1μM, orange fluorescent (540/560) 2% solids) in 100μL distilled water (solution 1) 0.2%

2) Prepare the following subsequent dilutions

  • solution 2: 10μL solution 1 in 100μL distilled water 0.02%
  • solution 3: 10μL solution 2 in 100μL distilled water 0.002%
  • solution 4: 10μL solution 3 in 100μL distilled water 0.0002%

Part 3: Preparing dilutions of dye containing hydrogels (marked with *)

1) Dilute 2mg hydrogels containing 30μL R6G (supernatant was previously removed) with 400μL phosphate buffer (solution 1)

  • Dilute 200μL of solution 1 with 200μL phosphate buffer (solution 2)
  • Dilute 200μL of solution 2 with 200μL phosphate buffer (solution 3)
  • Dilute 200μL of solution 3 with 200μL phosphate buffer (solution 4)
  • Dilute 200μL of solution 4 with 200μL phosphate buffer (solution 5)

2) Dilute 200μL 2mg of hydrogel solution containing dye in 1.5mL phosphate buffer (from 3/22/13) (marked with heart) with 200μL phosphate buffer (solution 1)

  • Dilute 200μL of solution 1 with 200μL phosphate buffer (solution 2)
  • Dilute 200μL of solution 2 with 200μL phosphate buffer (solution 3)
  • Dilute 200μL of solution 3 with 200μL phosphate buffer (solution 4)
  • Dilute 200μL of solution 4 with 200μL phosphate buffer (solution 5)

Part 4: Preparation of hydrogels

1) Prepare the following:

  • 2 samples of 0.5g (89,000-98,000 g/mol) PVOH in 6mL phosphate buffer (labelled A and B)
  • 2 samples of 0.5g (89,000-98,000 g/mol) PVOH in 6mL phosphate buffer with 300μL 1mM R6G (labelled C and D)

2) Heat solutions at ~110°C for 15 minutes. Allow to cool to room temperature.

3) mix with 40mL safflower oil for 1-2min on lower setting of blender

4) Freeze with liquid nitrogen and place in freezer at -20°C for 24 hours

Observations

  • FCS measurements were taken of the the 0.02% beads as well as solutions 2 and 5. According to our measurements, there were no spheres present in solutions 2 or 5. Because of this, we are going to prepare new sets of hydrogels (Part 4) in which we do not allow them to dry after filtration. We will instead immediately place them back into solution. We are hopeful that this will allow the hydrogels to stay separate instead of aggregating.




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