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RNeasy_Plus_Mini_Kit

RNeasy_Plus_Mini_Kit

Quick-Start Protocol

Sample & Assay Technologies

RNeasy ? Plus Mini Kit

The RNeasy Plus Mini Kit (cat. nos. 74134 and 74136) can be stored at room temperature (15–25°C) for at least 9 months.

For more information, additional and more detailed protocols, and safety information, please refer to the RNeasy Plus Mini Handbook , which can be found at http://www.wendangku.net/doc/1ee1b1156edb6f1aff001fdf.html/handbooks.

For technical assistance, please call toll-free 00800-22-44-6000, or find regional phone numbers at http://www.wendangku.net/doc/1ee1b1156edb6f1aff001fdf.html/contact.

Notes before starting

If purifying RNA from cell lines rich in RNases, or tissue, add either 10 μl β-mercaptoethanol (β-ME), or 20 μl 2 M dithiothreitol (DTT), to 1 ml Buffer RLT Plus before use. Buffer RLT Plus containing DTT or β-ME can be stored at room temperature for up to 1 month.

Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution.

Foaming can be reduced by adding Reagent DX (cat. no. 19088) at a final concentration of 0.5% (v/v) before disruption and homogenization.* * This option not included in handbook; handbook to be updated.

1. Cells : Harvest a maximum of 1 x 107 cells, either as a cell pellet, or lysed

directly in the vessel. Add the appropriate volume of Buffer RLT Plus (see Table 1). Vortex for 30 s, or homogenize.

Tissues : Disrupt the tissue (≤ 30 mg) and homogenize the lysate in the appropriate volume of Buffer RLT Plus (see Table 1). Centrifuge the lysate for 3 min at maximum speed. Carefully remove the supernatant by pipetting and use it in step 2.

2. Transfer the homogenized lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube (supplied).

3. Centrifuge for 30 s at ≥8000 x g (≥10,000 rpm). Discard the column, and save the flow-through. Add 1 volume (usually 350 μl or 600 μl) of 70% January 2011

RNeasy_Plus_Mini_Kit

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