BDtract TM Genomic DNA Isolation Kit Instruction Manual Catalog No. SA-40002: 50 preparation SA-40001: 100 preparation Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) 989-6296 / Fax: (650)871-2857 http://www.maximbio.com E-mail: mbi@maximbio.com
I. Intended Use For isolation and purification of genomic DNA from human whole blood, cultured cells tissue and bacteria cells. For Research Use Only. II. Introduction The BDtract TM Genomic DNA Isolation Kit is used to quickly and efficiently isolate high molecular weight (50-100 Kb in size) genomic DNA from whole blood, buffy coated lymphocytes, cultured cells, tissues and bacteria cells. This kit precludes the need for phenol, chloroform, or other organic extraction. The DNA is free of RNA, protein and degrading enzymes. The DNA product can be used for all molecular biology applications (RFLP, PCR amplification, etc.). This method is safe, fast and simple for DNA preparation from different sources of samples. The BDtract TM kit is available two sizes to suit your need: 50 and 100 preparations/kit. The protocol is based on a new method that simplified the purification process. It includes: 1. Lyse the whole blood cells or cytoplasma membrane with BD-1 solution. 2. Wash out the red blood cells with BD-2 solution. 3. Lyse the nucleate cells with anionic detergent BD-3 solution. 4. Remove the RNA contamination with RNase A solution. 5. Salt out protein debris with BD-4 solution. 6. Precipitate Genomic DNA with isopropanol and dissolve in TE buffer. III. 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. 1984. IV. Storage All components of this kit except RNase A solution can be stored at room temperature; stable at least for one year. RNase A solution should be stored at 4 C. V. Reagents Supplied Reagent/Cat. No. SA-40002 SA-40001 BD-1 Solution: 125 ml 250 ml BD-2 Solution: 125 ml 250 ml RNase A Solution: 0.5 ml 1 ml BD-3 Solution: 40 ml 80 ml BD-4 Solution: 15 ml 30 ml Sterile H 2 O: 15 ml 15 ml 2
Whole Blood DNA Protocol Samples may be fresh, or frozen. Store samples at 2-8 C for not more than 5 days prior to DNA isolation. 1. Collect 2.5 ml of whole blood with anti-coagulation agent (i.e. EDTA at concentration: 1 mg/ml blood; Also See Note) and transfer it to a 15 ml polypropylene centrifuge tube. 2. Add 2.5 ml of BD-1 solution and invert several times until completely mixed. 3. Centrifuge for 10 minutes at 2,200xg using a bench top centrifuge. 4. Slowly pour off the supernatant and save the nuclear pellet. 5. Add 2.5 ml of BD-2 solution to wash the pellet. Spin at 2,200xg for 10 minutes as before. 6. Discard the supernatant and gently resuspend the pellet with 0.6 ml of BD-3 solution by pipetting back and forth several times. Incubate at 65 C for 15 minutes. (Note: If a precipitate forms in BD-3 solution, warm up at 37 C before use) 7. Add 0.2 ml of BD-4 solution into the tube and mix well. 8. Spin for 10 minutes at maximum speed in a microcentrifuge. Carefully collect 9. Add 0.6 ml of Isopropanol the supernatant at room temperature. 10. Mix the sample by inverting the tube 40-50 times until the white threads of 11. Spin for 10 minutes at maximum speed in a microcentrifuge. 12. Discard the supernatant and wash DNA with 1 ml of ice-cold 70% ethanol. 13. Spin for 5 minutes at maximum speed in a microcentrifuge and discard the 14. Briefly dry the pellet and add 0.5 ml sterile H 2 O or TE buffer to the DNA. 15. Heat the tube at 65 C for 15-30 minutes and invert several times during the 16. Briefly spin down the DNA and use a aliquot to determine the DNA 17. It is ready to use. (Avoid to repeat pipetting and freezing /thawing the DNA.) Cell Culture DNA Protocol: Samples may be either fresh or frozen. Store samples at 2-8 C for not more than 5 days prior to DNA isolation. 1. Collect 2-4 million fresh cells by centrifugation at 1,500 xg for 5 minutes. (See Note) 2. Discard the supernatant and resuspend the pellet into 1.25 ml of BD-1 solution and transfer into a microcentrifuge tube. 3. Spin for 30 seconds at maximum speed in a microcentrifuge. Slowly pour off the supernatant and save the nuclear pellet. 3
4. Gently resuspend the nuclear pellet with 0.6 ml of BD-3 solution by pipetting back and forth several times. Incubate at 65 C for 15 minutes. (Note: If a precipitate forms in BD-3 solution, warm up at 37 C before use) 5. Cool down the solution to room temperature, and add 10 ul of the RNase A Solution to the cell lysate. 6. Incubate at 37 C for 15-30 minutes. 7. Keep the cell lysate on the ice for 5 minutes. 8. Add 0.2 ml of BD-4 solution into the tube and mix well. 9. Spin for 10 minutes at maximum speed in a microcentrifuge. Carefully collect 10. Add 0.6 ml of Isopropanol the supernatant at room temperature. 11. Mix the sample by inverting the tube 40-50 times until the white threads of 12. Spin for 10 minutes at maximum speed in a microcentrifuge. 13. Discard the supernatant and wash DNA with 1 ml of ice-cold 70% ethanol. 14. Spin for 5 minutes at maximum speed in a microcentrifuge and discard the 15. Briefly dry the pellet and add 0.2 ml sterile H 2 O or TE buffer to the DNA. 16. Heat the tube at 65 C for 15-30 minutes and invert several times during the 17. Briefly spin down the DNA and use a aliquot to determine the DNA 18. It is ready to use. (Avoid to repeat pipetting and freezing /thawing the DNA.) Bacteria Culture DNA Protocol: Samples may be either fresh or frozen. Collect overnight bacterial cultures and store on ice until use. 1. Add 1 ml cell suspension (overnight culture) to a 1.5 ml tube. 2. Centrifuge at 1,3000 xg for 5 seconds to pellet cells. 3. Discard the supernatant and resuspend the pellet into 1.25 ml of BD-1 solution. 4. Spin for 30 seconds at maximum speed in a microcentrifuge. Slowly pour off the supernatant and save the nuclear pellet. 5. Gently resuspend the nuclear pellet with 0.6 ml of BD-3 solution by pipetting back and forth several times. Incubate at 65 C for 15 minutes. (Note: If a precipitate forms in BD-3 solution, warm up at 37 C before use) 6. Cool down the solution to room temperature, and add 10 ul of the RNase A Solution to the cell lysate. 7. Incubate at 37 C for 15-30 minutes. 8. Keep the cell lysate on the ice for 5 minutes. 9. Add 0.2 ml of BD-4 solution into the tube and mix well. 10. Spin for 10 minutes at maximum speed in a microcentrifuge. Carefully collect 11. Add 0.6 ml of Isopropanol the supernatant at room temperature. 4
12. Mix the sample by inverting the tube 40-50 times until the white threads of 13. Spin for 10 minutes at maximum speed in a microcentrifuge. 14. Discard the supernatant and wash DNA with 1 ml of ice-cold 70% ethanol. 15. Spin for 5 minutes at maximum speed in a microcentrifuge and discard the 16. Briefly dry the pellet and add 0.2 ml sterile H 2 O or TE buffer to the DNA. 17. Heat the tube at 65 C for 15-30 minutes and invert several times during the 18. Briefly spin down the DNA and use an aliquot to determine the DNA 19. It is ready to use. (Avoid to repeat pipetting and freezing /thawing the DNA sample) Animal Tissue DNA Protocol: Samples may be fresh, frozen or fixed. Collect fresh animal tissue sample quickly and keep on ice at all times. 1. Add 10-20 mg of fresh or frozen tissue with chilled 0.6 ml of BD-3 solution and quickly homogenize about 30-50 strokes with microcentrifuge pestle. 2. Incubate lysate at 65 C for 15-60 minutes. (For maximum yield, add 3 ul Proteinase K Solution [20 mg/ml] to the lysate and incubate lysate at 55 C for 3 hours to overnight). 3. Cool down the solution to room temperature, and add 10 ul of the RNase A Solution to the cell lysate. 4. Incubate at 37 C for 15-30 minutes. 5. Keep the cell lysate on the ice for 5 minuets. 6. Add 0.2 ml of BD-4 solution into the tube and mix well. 7. Spin for 10 minutes at maximum speed in a microcentrifuge. Carefully collect 8. Add 0.6 ml of 100% ethanol the supernatant at room temperature. 9. Mix the sample by inverting the tube 40-50 times until the white threads of 10. Spin for 10 minutes at maximum speed in a microcentrifuge. 11. Discard the supernatant and wash DNA with 1 ml of ice-cold 70% ethanol. 12. Spin for 5 minutes at maximum speed in a microcentrifuge and discard the 13. Briefly dry the pellet and add 0.2 ml sterile H 2 O or TE buffer to the DNA. 14. Heat the tube at 65 C for 15-30 minutes and invert several times during the 15. Briefly spin down the DNA and use a aliquot to determine the DNA 16. It is ready to use. (Avoid to repeat pipetting and freezing /thawing the DNA sample) 5
Note: DNA extracted from the blood with heparin can influence treatment with restriction enzymes or PCR reactions. Average yield of genomic DNA prepared by this kit. Yield (ug) Whole Blood 1 ml 15-20 5 ml 100-125 Culture Cells 2 x 10 6 15-30 1 x 10 8 350-450 Tissue Culture 15 mg 15-20 100 mg 90-110 Bacteria 4 x 10 9 15-20 1 x 10 11 300-400 Reference: 1. Casareale D. et. al. (1992). PCR Methods and Applications 2: 149-153. 2. Everson R.B. et. al. (1993) BioTechniques 15: 18-20 3. Hanson C.A. and J.H. Kersey (1988) Amer. J. Hematology 28: 176-180. 4. Johns M.B. and J.E. Paulus-Thomas (1989). Anal. Biochem. 180: 276-278. 5. Lahiri D.K. and J. I. Nurnberger, Jr. Nucleic Acids Research 19: 5444 6. Lahiri D.K. et. al. (1992) J. Biochem. & Biophy. Methods 25: 193-205. 7. Miller S.A. et. al. (1988) Nucleic Acid Research 16: 1215. 8. Niku, S.D. et. al. (1987) J. Immun. Meth. 105: 9-14. 9. Parzer S. and Mannhalter C. (1991) Biochem. J. 273: 229-231. 6