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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 https://www.wendangku.net/doc/1617941399.html,/handbooks.

For technical assistance, please call toll-free 00800-22-44-6000, or find regional phone numbers at https://www.wendangku.net/doc/1617941399.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

Sample & Assay Technologies For up-to-date licensing information and product-

specific disclaimers, see the respective QIAGEN kit

handbook or user manual.

“RNA later ?” is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents.

ethanol to the flow-through, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 4.

4. Transfer up to 700 μl of the sample, including any precipitate, to an

RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid, and centrifuge for 15 s at ≥8000 x g . Discard the flow-through.

5. Add 700 μl Buffer RW1 to the RNeasy Mini spin column (in a 2 ml

collection tube). Close the lid, and centrifuge for 15 s at ≥8000 x g .

Discard the flow-through.

6. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g . Discard the flow-through.

7. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm).

Optional : Place the RNeasy spin column in a new 2 ml collection tube (supplied). Centrifuge at full speed for 1 min to further dry the membrane. 8. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50 μl RNase-free water directly to the spin column membrane. Close the lid, and centrifuge for 1 min at ≥8000 x g to elute the RNA.

Optional : Repeat elution with another volume of water or with RNA eluate. Table 1. Volumes of Buffer RLT Plus for sample disruption and

homogenization Sample Amount Dish Buffer RLT Plus*Disruption and homogenization

<5 x 106 <6 cm 350 μl Pelleted cells ≤1 x 107 6–10 cm 600 μl Add Buffer RLT Plus, vortex

(≤1 x 105 cells);

or use QIAshredder, TissueRuptor ?,

or needle and syringe

<20 mg – 350 μl Animal tissues 20–30 mg – 600 μl

TissueLyser LT; TissueLyser II;

TissueRuptor, or mortar and pestle

followed by QIAshredder

or needle and syringe