Fong:Quantification

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Overview

This procedure is used to quantify carbohydrates in solution. Primarily, this is used to observe cellobiose or glucose uptake rates of various organisms including C. thermocellum and T. fusca.

Materials

• Samples (
• 96-well PCR plate & cover
• 96-well Spectrophotometry plate
• Multi-channel pipette*
• Buffer
• DNS reagent (use gloves & lab coat)
• Prepared standard curve solutions

• PCR Thermocycler
• Multi-well spectrophotometer

*Not required, but certainly handy.

Procedure

1. Thaw samples and transfer 1 mL to a 1.5 mL centrifuge tube.
2. Return samples to -80°C freezer.
3. Spin down samples (max RPM for 3 minutes).
4. Add 20 μL of standard solution (in triplicate) to the 96-well PCR plate.
5. Add 20 μL H₂O in triplicate as a zero reference.
6. Add samples in triplicate.
7. Add 40 μL of buffer to each well used.
8. Add 120 μL of DNS reagent to each well.
9. Incubate plate at 95°C for 5 minutes (use thermocycler).
10. Cool on ice for ~5 minutes.