4110133B:4110133A.qxd 1/16/2009 2:35 PM Page COV2 Aurum™ Total RNA Mini Kit Instruction Manual Catalog # 732-6820 For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).
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4110133B:4110133A.qxd 1/16/2009 2:35 PM Page TOC1 Table of Contents Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Section 2 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .1 Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .2 Section 4 Necessary Supplies . . . . . . . . . . . . . . . . . . . . . .2 Section 5 Before Using the Aurum™ Total RNA Mini Kit . . . . . . . . . . . . . . . . . .3 Starting Material Amounts . . . . .
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4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 1 Section 1 Introduction The Aurum™ total RNA mini kit purifies total RNA samples rapidly from mammalian cell cultures, bacteria, and yeast (Saccharomyces cerevisiae), as well as animal and plant tissue. Total RNA samples prepared using the Aurum total RNA mini kit are suitable for use in a variety of downstream applications, including reverse transcription-PCR (RT-PCR), real-time PCR, and northern blots.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 2 Section 3 Storage Conditions All kit components (including lyophilized DNase I) should be stored at room temperature. Store reconstituted DNase I at –20°C in a nonfrost-free freezer, avoiding repeated freeze-thaw cycles. If precipitation is observed in any solution, warm the solution to 37°C to redissolve and allow the solution to return to room temperature before use.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 3 Section 5 Before Using the Aurum Total RNA Mini Kit Please read the following guidelines before proceeding with the total RNA purification. Starting Material Amounts The Aurum total RNA mini kit is designed to process up to the amounts indicated below (per column): • 2 x 106 mammalian cultured cells • 3 OD•ml* of gram-positive or gram-negative bacteria 3 OD•ml of bacteria roughly corresponds to a culture volume of 500–750 µl • 3 OD•ml of yeast (S.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 4 Table 1. Yield (per column) of total RNA from various samples using the Aurum total RNA mini kit. Starting Material Avg. Yield (µg)* 6 Cultured cells (2 x 10 ) 3T3 HeLa 20–30 20–40 Bacteria (2.4 x 109) E. coli B. cereus 30–35 30–35 Yeast (3 x 107) S.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 5 Reagents Used With the Aurum Total RNA Mini Kit • The low stringency wash solution is provided as a 5x concentrate. Add 4 volumes (80 ml) of 95–100% ethanol to the low stringency wash solution concentrate before initial use • Before using the lysis solution, add 500 µl of b-mercaptoethanol (catalog # 161-0710) to the solution for a final concentration of 1% • The RNase-free DNase I is provided as a lyophilized powder.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 6 Elution Guidelines • Apply elution solution directly to the membrane stack at the base of each RNA binding column Ribonucleases • Although the components of this kit are provided free of contaminating ribonucleases, great care must be taken not to contaminate the solutions or the RNA binding columns. Gloves should always be worn when handling RNA and should be changed frequently.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 7 Table 2. Disruption and homogenization methods.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 8 Section 6 Vacuum Manifold Setup and Use With the Column Adaptor Plate (CAP) Guidelines for Vacuum Format • The recommended operating range is –17 to –23 inHg. Do not exceed –25 inHg when performing this protocol. A vacuum regulator is strongly recommended to establish the appropriate negative pressure Table 3. Pressure unit conversions. To convert from inches of mercury (inHg) to: millimeters of mercury or torr (mmHg, torr) millibar (mbar) Multiply by: 25.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 9 Preparing the Aurum Vacuum Manifold Tubing provided in the Aurum vacuum manifold kit (catalog # 732-6470) is 4 ft long and must be cut into appropriate pieces before proceeding. Prior to setup, you may ensure that the gauge pointer is adjusted to zero by removing the lens cover, followed by turning the adjustment pin located beneath the dial face. Vacuum Setup (Figure 1) 1. Cut tubing into three pieces of appropriate length. 2.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 10 Manifold Wash Setup (Figure 2) 1. Insert the CAP (luer ends up) into the depression in the vacuum manifold top. Ensure that the CAP rests evenly on the gasket. 2. Insert the luer ends of the desired columns into the available luer fittings, ensuring a tight fit. 3. Close the unused luer fittings with the caps provided. Close caps by rotating clockwise until light resistance is encountered.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 11 Section 7 Vacuum Protocol Important: Please read Section 5, “Before Using the Aurum™ Total RNA Mini Kit” and Section 6 "Vacuum Manifold Setup and Use With the Column Adaptor Plate (CAP)," before proceeding. This procedure requires the Aurum vacuum manifold and column adaptor plate (catalog # 732-6470), or any vacuum manifold with luer fittings. Vacuum filtration steps should be carried out at –17 to –23 inHg for optimum performance.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 12 Bacteria Follow steps B1–B4, then continue with step 1 of “All Starting Sample Types” on page 13. B1. Transfer up to the equivalent of 3 OD•ml bacterial culture into a 2 ml capped microcentrifuge tube (provided). Centrifuge at maximum speed for 1 min. Decant the supernatant and blot the tube with paper towels. B2. Add 100 µl of 500 µg/ml lysozyme in TE (10 mM Tris, 1 mM EDTA, pH 7.5) to each tube. Pipet up and down to resuspend the pellet thoroughly.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 13 Animal and Plant Tissue Follow steps D1–D5, then continue with step 1 of "All Starting Sample Types" on page 13. D1. Cut the tissue into small pieces (<5 mm long) and grind it into a fine powder with a mortar and pestle containing liquid nitrogen. Make sure that the tissue does not thaw by periodically adding liquid nitrogen to the mortar. D2.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 14 4. Add 700 µl of low stringency wash solution to the RNA binding column and close the vacuum regulator dial until the gauge indicates –17 to –23 inHg. Continue to apply the vacuum until the low stringency wash solution has passed through the column. Open the vacuum regulator until the gauge indicates 0 inHg. 5. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute the DNase I by adding 250 µl 10 mM Tris, pH 7.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 15 Section 8 Spin Protocol Important: Please read Section 5, “Before Using the Aurum™ Total RNA Mini Kit,” before proceeding. The Aurum total RNA mini kit can be used with any commercially available microcentrifuge that can accommodate 1.5 and 2.0 ml tubes. All centrifugation steps are performed at maximum speed (>12,000 x g) at room temperature. Note: Except for the first few steps that are specific for the starting sample types (A.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 16 B3. Add 350 µl of lysis solution (already supplemented with 1% b-mercapto-ethanol) to each tube. Pipet up and down several times to mix thoroughly. B4. Add 250 µl of 70% isopropanol (not supplied) to each tube. Pipet up and down to mix thoroughly. Make sure that no bilayer is visible and that the viscosity is substantially reduced. Yeast Follow steps C1–C6, then continue with step 1 of “All Starting Sample Types” on page 17. C1.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 17 D2. Transfer up to 20 mg hard animal tissue, up to 40 mg soft animal tissue, or up to 60 mg plant tissue into an RNase-free 2.0 ml capped microcentrifuge tube (provided). Let the tissue thaw before adding the lysis solution or precipitation may occur. Note: Plant tissue requires the Aurum lysis solution to be supplemented with 2% (w/v) polyvinylpyrrolidone-40 (PVP).
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 18 6. For each column processed, mix 5 µl of reconstituted DNase I with 75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube (not provided). Scale up proportionally if processing more than one column. Add 80 µl of diluted DNase I to the membrane stack at the bottom of each column. Allow the digest to incubate at room temperature for 15 min (25 min for animal tissue). 7. Add 700 µl of high stringency wash solution to the RNA binding column.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 19 Section 9 Troubleshooting Guide Problem Possible Cause Recommended Solution Genomic DNA contamination Incomplete DNase I digest Increase DNase I digest time Inactive DNase I Store reconstituted DNase I in a nonfrostfree freezer. Avoid freeze-thaw cycles.
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4110133B:4110133A.qxd 1/16/2009 2:35 PM Page COV1 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA (510) 741-1000 1-800-424-6723 Bio-Rad Laboratories, Inc. Web site www.bio-rad.
