Sorbitol/trehalose stock solution
MATERIALS
Reagents
Prepare all reagents with nuclease-free H2O.
Chelex 100 resin (Sigma C7901)
D-Sorbitol (Wako 198-03755) (0.89 g/mL [4.9 M])
Autoclave for 30 min at 121°C.
D-(+)-Trehalose dihydrate (Fluka/Sigma-Aldrich 90208) (0.727 g/mL)
Autoclave for 30 min at 121°C.
Equipment
Centrifuge, benchtop (e.g., Allegra X-12R; Beckman Coulter)
Centrifuge tubes (15-mL)
Pipettes, disposable
Vortex mixer
METHOD
Perform all procedures under RNase-free conditions.
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1. Add 5 mL of the saturated trehalose solution to a tube containing 10 mL of 4.9 M sorbitol solution.
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2. Using a disposable pipette, add <1 cm of Chelex 100 resin to the tube. Vortex well.
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3. Incubate the solution for 3 h at room temperature (25°C) without further agitation.
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4. Centrifuge at 2000 rpm for 10 min.
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5. Transfer the supernatant to a fresh tube. Store for up to 6 mo at -20°C.
The final solution is 3.3 M sorbitol/0.66 M trehalose.
Solutions deionized with Chelex 100 can turn yellow even after centrifugation, but this does not affect their use in nanoCAGE library production.
