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10001481A.qxp 3/7/2005 7:05 AM Page i Table of Contents Section 1 Introduction ...........................................................1 Kit Components.................................................................2 Storage and Stability .........................................................2 Special Handling Instructions............................................2 Section 2 Section 3 Materials and Equipment Required (Not Provided) .......................................................
10001481A.qxp 3/7/2005 7:05 AM Page 1 Section 1 Introduction PureZOL RNA isolation reagent is intended for the extraction of total RNA from animal and plant tissues, cultured mammalian cells, and bacterial and yeast cells in under 1 hour. PureZOL can also be used for the simultaneous extraction of RNA, DNA, and proteins from various samples.
10001481A.qxp 3/7/2005 7:05 AM Page 2 Kit Components Catalog # 732-6890 – PureZOL RNA isolation reagent, 100 ml Storage and Stability PureZOL RNA isolation reagent is shipped at room temperature. This product is guaranteed for 12 months from the date of purchase when stored at 2–8°C and kept away from light. Special Handling Instructions PureZOL RNA isolation reagent contains a poison (phenol) and an irritant (guanidine thiocynate). The reagent causes burns and can be fatal if ingested.
10001481A.qxp 3/7/2005 7:05 AM Page 3 Section 2 Materials and Equipment Required (Not Provided) Microcentrifuges (capable of >12,000 x g) — at 4°C and at room temperature 95–100% ethanol, ACS grade or better Chloroform (without additives such as isoamyl alcohol); need 0.2 ml of chloroform per 1 ml of PureZOL Isopropyl alcohol; need 0.
10001481A.qxp 3/7/2005 7:05 AM Page 4 Section 3 Maintaining an RNase-Free Environment To avoid introducing RNases, great care must be taken in handling the reagents and purified RNA samples. Care must be taken to proceed through the RNA isolation as quickly as possible. An RNase-free environment will yield the best results. If possible, work in an RNase-free environment; use latex or vinyl gloves when handling reagents or RNA and change gloves frequently.
10001481A.qxp 3/7/2005 7:05 AM Page 5 Solutions that are prepared by the user should be treated with DEPC to inactivate RNases. See instructions below on how to prepare DEPC-treated solutions. Keep reagent bottles closed when not in use and keep RNA samples on ice to prevent degradation by RNases. Preparing Reagents Not Supplied With the Kit Note: DEPC is destroyed by primary amines. If a solution containing a primary amine will be DEPC-treated, omit the amine in preparing the solution.
10001481A.qxp 3/7/2005 7:05 AM Page 6 Section 4 Recommendations for Best Results • When isolating RNA from small sample sizes (<500,000 cells or <10 mg of tissue), lyse or homogenize in 0.8 ml of PureZOL. Use glycogen as an RNA carrier by adding 5 µl of a 20 mg/ml glycogen solution (not provided) to the aqueous phase before precipitation with isopropyl alcohol. Carry out precipitation for 30 minutes at 4°C.
10001481A.qxp 3/7/2005 7:05 AM Page 7 Section 5 Protocol Carry out all steps at room temperature unless otherwise indicated. RNase-free disposable polypropylene tubes should be used throughout the procedure. The entire procedure should take less than 1 hour. 1. Disrupt and homogenize the sample using the following suggestions depending on the sample type: Cells Grown in a Monolayer Cells grown in a monolayer should be lysed with PureZOL directly in the culture dish.
001481A.qxp 3/7/2005 7:05 AM Page 8 recommended to disrupt the cell walls of yeast and bacteria. Bacteria and yeast lysate can also be heated to 55°C for 10 minutes prior to adding chloroform to increase the effectiveness of lysis by PureZOL. Note: Do not wash cells prior to the addition of PureZOL as this could increase the possibility of mRNA degradation. Fresh Tissue Freshly harvested tissue samples should be processed immediately after dissection to avoid RNA degradation.
10001481A.qxp 3/7/2005 7:05 AM Page 9 100 mg of tissue and transfer the sample into a suitable sized tube for disruption and homogenization. Add 1 ml of PureZOL reagent to every 50–100 mg of the frozen ground tissue and immediately homogenize for 30–60 seconds using a rotor-stator or bead mill homogenizer (refer to manufacturer's instructions for details).
10001481A.qxp 3/7/2005 7:05 AM Page 10 top layer. Carryover of the solid debris can cause column clogging and affect RNA sample purity. 3. Add 0.2 ml of chloroform per 1 ml of PureZOL used in step 1, then cover and shake vigorously for 15 seconds. Do not vortex. 4. Incubate for 5 minutes at room temperature while periodically mixing the sample. 5. Centrifuge at 12,000 x g for 15 minutes at 4°C.
10001481A.qxp 3/7/2005 7:05 AM Page 11 8. Centrifuge at 12,000 x g for 10 minutes at 4°C. 9. The RNA will appear as a white pellet on the side and bottom of the tube. Carefully discard the supernatant. 10.To wash the RNA pellet, add 1 ml of 75% ethanol for every 1 ml of PureZOL used in step 1. At this point, the sample can be stored in ethanol at 4°C for at least 1 week or at -20°C for at least 1 year. 11.Vortex the sample and then centrifuge at 7,500 x g (max) for 5 minutes at 4°C.
10001481A.qxp 3/7/2005 7:05 AM Page 12 Section 6 Troubleshooting Problem Possible Cause Recommended Solution Incomplete separation of phases after centrifugation Lysate was not mixed properly after adding chloroform (See step 3 in protocol) Once chloroform is added, mix tubes vigorously by shaking for 15 seconds. Do not vortex.
10001481A.qxp Problem 3/7/2005 7:05 AM Page 13 Possible Cause Recommended Solution Low yield (continued) the pellet. If necessary, incubate the resuspended pellet for 10 minutes at 55–60°C and then pipet up and down Low amount of starting material When isolating RNA from small sample amounts, homogenize the sample in 0.8 ml of PureZOL. Use glycogen as an RNA carrier by adding 5 µl of a 20 mg/ml glycogen solution (not provided) to the aqueous phase before precipitation with isopropyl alcohol.
10001481A.qxp 3/7/2005 7:05 AM Page 14 Problem Possible Cause Recommended Solution Low yield (continued) Insufficient amount of PureZOL used for sample lysis and homogenization Scale up the amount of PureZOL used according to sample size.
10001481A.qxp 3/7/2005 7:05 AM Page 15 Problem Possible Cause Recommended Solution RNA is degraded (continued) Cells grown in either monolayer or suspension were washed prior to homogenization with PureZOL For cells grown in monolayer, aspirate the growth medium and then add PureZOL directly to the plate. No washing or trypsinization is necessary.
10001481A.qxp 3/7/2005 7:05 AM Page 16 Problem Possible Cause Recommended Solution Genomic DNA contamination (continued) Phase separation not performed at the right temperature Make sure that centrifugation step is performed at 4°C following the addition of chloroform in order to achieve complete separation of the phases Insufficient amount of PureZOL used for sample lysis and homogenization Scale up the amount of PureZOL used according to sample size.
10001481A.qxp 3/7/2005 7:05 AM Page 17 Section 8 Ordering Information Catalog # Description 732-6890 PureZOL RNA isolation reagent, 100 ml Related Products Aurum Total RNA Kits for Isolation of High Quality RNA From a Variety of Samples 732-6820 Aurum Total RNA Mini Kit, 50 preps, includes 50 RNA binding columns, 50 capless collection tubes (2.0 ml), 100 capped microcentrifuge tubes (2.0 ml), 50 capped microcentrifuge tubes (1.
10001481A.qxp 3/7/2005 7:05 AM Page 18 Legal Notices PureZOL RNA isolation reagent is subject to US Patent 5,346,994. Speed Vac is a trademark of Sevant Instruments, Inc.
10001481A.qxp 3/7/2005 7:05 AM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
