Aurum Total RNA 96 Kit ™ Instruction Manual Catalog #732-6800
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 96 Kit . . . . . . . . . . . . . . . . . . . 3 Starting Material Amounts . . . . . . . . . . . . . . . . . . . . . . .
Section 1 Introduction The Aurum™ Total RNA 96 kit rapidly purifies up to 192 total RNA samples from biological samples (e.g. mammalian cells, yeast, or bacteria) with minimal handling. Total RNA samples prepared using the Aurum™ Total RNA 96 kit are suitable for use in a variety of downstream applications, including reverse transcription-PCR (RT-PCR), quantitative real-time PCR, microarray analysis, and northern blots.
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 nonfrostfree 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.
Section 5 Before Using the Aurum™ Total RNA 96 Kit Please read the following guidelines before proceeding with the total RNA purification.
Table 1. Yield (per well) of total RNA from various samples using the Aurum™ Total RNA 96 kit. Starting Material Avg. Yield (µg)* Cultured cells (1 x 10 ) 3T3 7–10 HeLa 11–17 6 Bacterial (8 x 108) E. coli 5 B. cereus 5 Yeast (2 x 107) S. cerevisiae 9–11 Starting material amounts in parentheses are the maximum amounts recommended for use with the Aurum™ Total RNA 96 kit. *Yield figures are representative of a minimum of four full plate experiments.
• V endors of lyticase, which is used to partially degrade the cell walls of yeast cells, may have different definitions of the enzyme’s activity. As used in this instruction manual, 1 unit of lyticase produces a ∆A800 of 0.001/min at pH 7.5 at 25°C, using 3 ml of yeast suspension as a substrate in a 3 ml reaction volume. Elution Guidelines • A pply elution solution directly to the membrane stack at the base of each RNA binding plate.
Disruption and Homogenization Proper disruption and homogenization of the starting materials are required to ensure complete lysis of the cells and to reduce the viscosity of the cell lysates. This procedure uses repeated pipetting up and down through micropipettor tips to aid in lysing the cells, as well as to prepare homogeneous lysates prior to loading onto the RNA binding plate. Make sure that cell lysates are thoroughly homogenized (i.e.
Preparing the Aurum™ Vacuum Manifold Tubing provided in the Aurum™ Vacuum Manifold kit is 4 ft long and must be cut into appropriate pieces before proceeding. Prior to setup, you may ensure that the gauger pointer is adjusted to zero by removing the lens cover, followed by turning the adjustment pin located beneath the dial face. Vacuum Setup (Figure 2) 1. Cut tubing into three pieces of appropriate length. 2.
2. Place the desired 96-well binding plate on the manifold top and apply the recommended vacuum pressure for your application. Manifold Elution Setup (Figure 3B) 3. When eluting from the purification plate, place the stage on the manifold base, with the etched “This Side Up” facing up. Place a clean 96-well microtiter collection plate securely on top of the stage and replace the manifold top.
Section 7 Vacuum Protocol Please read Section 5, “Before Using the Aurum™ Total RNA 96 Kit” and Section 6, “Vacuum Manifold Setup and Use With 96-Well Plates” before proceeding. Except for the first few steps that are specific for the starting sample types (A, for cultured cell lines; B, for bacteria; and C, for yeast), the remaining procedures within “All Starting Sample Types” share a common protocol.
Yeast Follow steps C1–C5, then continue with step 1 of “All Starting Cell Types” on page 10. If starting with a grow block of yeast culture (maximum 2 OD/ ml/well), centrifuge the grow block at 1,500 x g for 10 min. Decant the supernatant, and blot the block with paper towels. C1. Prepare 100 ml lyticase dilution buffer: 1 M sorbitol 0.1 M EDTA, pH 7.4 0.1% (v/v) b-mercaptoethanol Equilibrate the buffer at 30°C before use. C2.
5. dd 700 µl of low stringency wash solution to each well of the RNA A binding plate. Gradually increase the negative pressure between –17 to –23 inHg over a 5–10 sec period by slowly closing the vacuum regulator. After all wells have emptied, open the vacuum regulator until the gauge reads approximately 0 inHg. 6. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not supplied) to the vial. Pipet up and down briefly to mix. 7.
Note: Gradual application of negative pressure is required to prevent sample spraying and cross-contamination. The eluted total RNA samples in the sample collection plate can be used immediately in downstream applications. Alternatively, the sample collection plate can be sealed with sealing tape and stored at –20°C or –80°C for later use. Section 8 Spin Protocol Please read Section 5, “Before Using the Aurum™ Total RNA 96 Kit” before proceeding.
B2. A dd 350 µl of lysis solution (already supplemented with 1% b-mercaptoethanol) to each sample and pipet up and down several times to mix thoroughly. B3. A dd 250 µl of 70% isopropanol (not supplied) to each sample and 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–C5, then continue with step 1 of “All Starting Cell Types” on page 13.
5. dd 700 µl of low stringency wash solution to each well of the RNA A binding plate. Centrifuge for 2 min at 1,500 x g. Discard the low stringency wash solution from the waste tray and replace the RNA binding plate on top of the same waste tray. 6. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not supplied, see note below) to the vial. Pipet up and down briefly to mix. 7. each well (of a 96-well plate) processed, mix 2.
Section 9 Troubleshooting Guide Problem Possible Cause Recommended Solution Difficulty achieving Purge step in protocol –17 to –23 inHg negative pressure If sealing of wells with sealing tape restores negative pressure, no corrective action is required; however, do not leave sealed Open vacuum regulator Turn control knob fully clockwise to close regulator Plate not seating on vacuum manifold Press down gently on RNA binding plate to seat Manifold top no
Problem Possible Cause Recommended Solution Low or highly variable Elution solution applied Apply elution solution eluate volumes among to RNA binding plate well directly to membranes at wells walls base of each well RNA binding plate not seated properly Set plate properly and press down gently to seat Abrupt application of negative pressure during elution Increase negative pressure gradually over 5–10 sec using the vacuum regulator Residual wash buffer on drip director
Problem Possible Cause Recommended Solution Clogging of RNA binding plate Excessive amount of starting material per well Reduce volume of culture used Incomplete mixing of lysis and alcohol solutions Mix RNA lysis solution and alcohol thoroughly by pipetting up and down Pipette lysate up and down further to reduce lysate viscosity Incomplete homogenization of cell lysate Incomplete digestion with lysozyme or lyticase Low RNA yield Low amount of starting
Problem Solution Possible Cause Recommended Total RNA prep Incorrect use of wash Add the appropriate performs poorly in solutions volume of 95–100% downstream ethanol to the low applications stringency wash solution before initial use RNA is degraded See problem “RNA degradation” Add 1–3 min to the purge time after the final wash step Ethanol contamination in prep (eluate volumes >60 µl) Blot RNA binding plate drip directors with paper towels; ensure that the plate
Section 10 Ordering Information Catalog # Description 732-6800 Aurum™ Total RNA 96 Kit 732-6470 Aurum™ Vacuum Manifold 732-6820 Aurum™ Total RNA Mini Kit 732-6830 Aurum™ Total RNA Fatty and Fibrous Tissue Kit 732-6870 Aurum™ Total RNA Fatty and Fibrous Tissue Module (without PureZOL™ RNA isolation reagent) 19
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