Catalog No. SA-40004: 50 preparation SA-40003: 100 preparation

Transcription

1 BDtract TM RNA/DNA Isolation Kit Instruction Manual Catalog No. SA-40004: 50 preparation SA-40003: 100 preparation Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) / Fax: (650) mbi@maximbio.com

2 Introduction The analysis of RNA and DNA from biological samples has become more and more important. However, the amount of biological samples is often limited. The RDtract TM kit is designed for simultaneous isolation of both RNA and DNA from one sample. The ability to extract RNA in an undegraded form from cells is of fundamental importance in molecular biology and is frequently one of the most frustrating procedures to perform successfully. Most of the commonly used procedures for RNA extraction are time consuming. This critical issue has been solved by the single-step method that allows preparation of pure RNA within one to two hours. The RDtract TM RNA/DNA ISOLATION KIT is used to simultaneously isolate RNA and DNA from tissue cultured cells, blood and bacteria. The method is simple and quick, particularly valuable when a large number of RNA/DNA samples are to be isolated. The extracted RNA is free of DNA, proteins and degrading enzymes, without the use of phenol, guanidine, resins or glass beads. It can be used for Northern blot analysis, RNase protection assays, RT-PCR, and Poly (A) + selection. The genomic DNA yielded by this procedure is greater than 50 Kb in length and free of RNA and proteins. It is ready for Southern hybridization and PCR amplification. For Research Use Only. Storage All components of this kit are stable at least for one year when stored at room temperature. Warnings and Precautions 1. Certain chemical reagents used in this kit, including Tris(hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) may be hazardous. They may be harmful if swallowed and contact with eyes and skin should be avoided. In case of contact wash with large amounts of water and seek medical attention. 2. Potential Biohazardous Materials: The blood and body fluids of all human subjects are considered potentially infectious for blood-borne pathogens. These samples should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control / National Institute of Health manual, Biosafety in Microbiological and Biomedical Laboratories Reagents Supplied Reagent/Cat. No. SA SA RS1 50 ml 100 ml RS2 15 ml 30 ml DS1 100 ml 200 ml DS2 10 ml 20 ml DEPC-H 2 O 5 ml 5 ml Reagents Needed: Chloroform (ACS grade) Isopropanol (ACS grade) 75% Ethanol (ACS grade) 2

3 Basic Protocol: RS1 Lysis Reagent RS2 RNA Precipitate Reagent DS1 Reverse Extraction Reagent for DNA DS2 DNA Precipitate Reagent Procedure: Note: All centrifugation should be performed at 10,000 to 12,000 xg. Microcentrifugation should be performed at the maximum speed. Part I - Extraction of RNA A. Cultured cells grown in suspension: Cells are sedimented at 1,000 xg for 5-10 minutes and the cell pellet is lysed in the RS1 (500 µl per 1-2 X 10 6 cells: Note 3). B. Cultured cells grown in monolayer: Cells are lysed directly in the culture dish in the RS1 (500 µl per well, 6-well culture dish). C. Blood: ( Note 4) Add 400 µl of RS1 into 100 µl whole blood and mix well by inverting the tube several times. D. Bacteria: Spin down 0.5 ml of overnight bacterial culture at 11,000 xg for 20 seconds and the cell pellet are lysed into 500 µl of RS1. General Protocol: 1. Pass cell lysate a few times through a pipette. 2. Add 150 µl cold RS2 and mix well by inversion several times. 3. Add 350 µl of Chloroform, mix and place on ice for 5 minutes. (Very important, see Note 5 for details) 4. Centrifuge the mixture for 5 minutes. 5. Transfer the aqueous phase (upper phase) to a fresh tube, add 650 µl icecold isopropanol. (Save lower organic phase for further DNA isolation). 6. Centrifuge RNA samples for 15 minutes at 4 C. RNA precipitate (often invisible before centrifugation) forms a white pellet at the bottom of tube. 7. Remove the supernatant and wash the RNA pellet with 75% ethanol. 8. Dry briefly the RNA pellet under vacuum for 5-10 minutes (It is important not to let the RNA pellet dry completely, as it will greatly decrease its solubility). 9. Dissolve the RNA pellet with DEPC-treated H 2 O. Part II - Recovery of DNA: 1. Return to the tube containing organic material saved from Part I, step 5. Remove any remaining aqueous material as completely as possible. 2. To the remaining organic/interface material, add an equal volume of DS1 and mix thoroughly by gentle shaking until the interphase turn into a white homogeneous liquid. Do not vortex. 3. Centrifuge for 10 minutes at 4 C. Transfer the aqueous phase (DNA) to a fresh tube and keep it on ice. 3

4 4. Repeat step Add equal volume of chloroform to each DNA aqueous sample. Mix well and gently. 6. Centrifuge for 5 minutes at 4 C. Transfer the aqueous to a fresh tube. 7. Add 1/10 volume of DS2 and equal volume of Isopropanol. Mix well and keep at -80 C for one hour of -20 C for overnight. 8. Centrifuge for 15 minutes at 4 C and discard the supernatant. Wash the DNA pellet with cold 75% ethanol and dry the pellet briefly under vacuum. Resuspend the DNA µl TE Buffer or H 2 O. Notes: 1. The final preparation of undegraded RNA is free of DNA and proteins and has an A 260 /A 280 ratio of Yield from 10 6 cultured cells = µg. The final preparation of DNA is free of RNA and proteins and has an A 260 /A 280 ratio of yield from 10 6 cultured cells = 4-8 µg. 2. Hands and dust may be the major source of RNase contamination. Use gloves and keep tubes closed. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. 3. The procedure is optimized for isolating RNA from 2 X 10 6 cultured cells or less amounts. For larger amounts of cultured cells or tissues, adjust the volumes of the reagents according to the table below: Cultured Cells Multiple Volume by 3-4 X X X X This kit can only isolate RNA from the blood but not simultaneously isolate the DNA from the blood. 5. Failure to incubate on ice at this step may result in low 260/280 ratio or genomic DNA contamination in the samples. Total RNA isolated from culture cells. Lane 1: 5 ug total RNA from Hela cells. Lane 2: 5 ug total RNA from P388D-1 cells. Lane 3: 5 ug total RNA from Jurket cells. Lane 4: 5 ug total RNA from Caski cells. Lane 5: 5 ug total RNA from SiHa cells. DNA isolated from culture cells. Lane 1: DNA from Hela cells. Lane 2: DNA from P388D-1 cells. Lane 3: DNA from Jurket cells. Lane 4: DNA from Caski cells. Lane 5: DNA from SiHa cells. 4

5 References: 1. Chattopadhyay, N., R. Kher and M. Godbole. (1993). Inexpensive SDS/phenol method for RNA extraction from tissues. BioTchniques 15: Chirgwin, J.M., A.E. Przbyla, R.J. Mae Donald and W.J. Rutter. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: Chomczynski, P. and N. Sacchi. (1987). Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162: Cox, R.A. (1968). The use of guanidium chloride in the isolation of nucleic acid. Methods Enzymol. 12: Farrell, R. E. Jr. RNA Methodologies: A laboratory Guide for Isolation and Characterization. Chapter Maniatis, T., E.F. Fritsch and J. Sambrook. (1989). Molecular Cloning: A Laboratory Press, Cold Spring Harbor, NY. 7. Salvatori, R., D. Primorac and A.C. Lichtler. (1994). An efficient procedure for separate extraction of nucleic and cytoplasmic RNA from cell culture. BioTechniques 16: