Filters and solutions

• Filters: protein-adsorbent membrane filters 25 mm diameter, 0.8 jjum pore size can be obtained from Gelman Sciences Inc. (Ann Arbor, MI) [PVC (polyvinyl chloride)/acrylic co-polymers, Metricel®] or from Poretics Corporation (Livermore, CA) (PVC).

• EDTA washing solution pH 10.0:

(a) Prepare Na2EDTA (0.1 M) from 37.2 g Na2EDTA.2H20(ethylene-diaminetetraacetic acid disodium salt: dihydrate) and 8 g NaOH made up to 1 litre with H20. Adjust pH to 10.0 with NaOH (5 M).

(b) For 500 ml of 0.02 M EDTA, mix 100 ml of Na2EDTA (0.1 M) and 400 ml of H20 and adjust the pH to 10.0.

• LS-10 (lysis solution pH 10.0): add 116.8 g NaCl, 400 ml 0.1 M Na2EDTA,

Figure 1. Photograph of a filter elution assay set-up, including a custom-designed rack to hold elution funnels and scinitillation vials. Front right: a rubber stopper with central needle (see text); front midle: the same with the assembled lower part of a Swinnex filter holder; front left and second row: four assembled elution funnels.

Figure 1. Photograph of a filter elution assay set-up, including a custom-designed rack to hold elution funnels and scinitillation vials. Front right: a rubber stopper with central needle (see text); front midle: the same with the assembled lower part of a Swinnex filter holder; front left and second row: four assembled elution funnels.

6.7 ml of 30% sarkosyl solution, and distilled water to a linal volume of 1 litre. Adjust to pH 10.0 with NaOH/HCl.

• nucleus buffer: add 0,136 g KH2P04, 1.016 g MgCb., 8.76 g Nad, 0.380 g ECjTA, and distilled water to a final volume of 1 litre. Adjust the pH to 6.4 with NaOH/HCl. Add DTT (dithiothrcitol) to 0.1 mM final concentration immediately before use.

• nucleus buffer + Triton X-100: add 1 ml of Triton X-100 (30% stock solution) to 100 ml of nucleus buffer.

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