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Synthesis of Carboxymethyl Reduced Dextran

Reagents:

  • Dextran T-10
  • Sodium Borohydride
  • Hydrochloric Acid (6M)
  • Sodium Hydroxide (8M for 2-Step 1:1 for 1-Step)
  • Bromoacetic Acid

Materials/Equipment:

  • Balance
  • Weigh boats
  • 500 ml auto-cleavable screw top bottle and magnetic stir bar
  • Ice (depending on desired production temp)
  • Hot plate with stirring
  • 50 ml graduated cylinder
  • 10 ml pipettes
  • 1000 ul pipette
  • 50 ml centrifuge tubes for reagent aliquots and storage
  • 100 ml beaker
  • Thin plastic bucket used for ice bath
  • Thermometer
  • Concentrator with 3 kDa membrane
  • pH meter
  • pH strips

2-Step Procedure:

Reduced Dextran:
  1. Dissolve 10g Dextran T-10 in 100 ml of Milli-Q water to a 500 ml screw top bottle at 25°C.
  2. Add 1 g of Sodium Borohydride and begin stirring with magnetic stir bar.
  3. Rest cap on top (leave small gap for gas release) and let stir for 12 hrs.
  4. Adjust pH to 5.0 with 6M HCL using pH meter to check progress (double check pH with pH strips).
  5. Concentrate solution down using 3 kDa filter using four 500 ml washes (make sure volume is below 100 ml before adding next wash.
    1. Can also Dextran Precipitation Purification Protocol
  6. Collect solution at approx. 50 ml.
CMRD:
  1. Add 32.5 ml of reduced dextran solution containing approx. 10 g reduced dextran/100 ml to a screw cap bottle with stir bar.
  2. Add 36 ml of 8M NaOH.
  3. Make solution of bromoacetic acid using 0.5525 g bromoacetic acid with 1.5 ml of MIlli-Q water.
  4. Add to dextran solution and stir at room temp for 1 hr.
  5. Add approximately 37.5 ml of crushed to the solution.
  6. Prepare ice bath for screw cap bottle.
  7. Adjust pH to 6.0 using 6M HCL and pH meter, ensuring temp does not go above room temp (double check pH with pH strips)
  8. Concentrate as done in steps 5 and 6 above.

1-Step Protocol:

  1. Add 0.4 g Sodium Borohydride to a screw top bottle with stir bar.
  2. Add 0.5 g of 1:1 Sodium Hydroxide solution.
  3. Prepare 25 g of Dextran T-10 into 50 ml Milli-Q water.
  4. Add Dextran solution to screw top bottle and stir at room temp for 4 hours (leave small gap for top to allow for gas release)
  5. Add 19.5 g of 1:1 NaOH solution
  6. Put screw top bottle on ice bath and add 6.2 g of bromoacetic acid (keep temp below room temp)
  7. Let solution stir for 16 hrs with cap slightly off.
  8. Adjust pH to 6.2 with 6M HCL on ice bath.
  9. Concentrate with 5 kDa membrane as listed in 2-Step reduced dextran protocol steps 5 and 6.