First-Beer INSTITUT FÜR ANGEWANDTE LABORANALYSEN GMBH First-Beer Magnetic DNA Kit Extraktion von Hefe- und Bakterien-DNA aus Bier und anderen Getränken Extraction of bacteria and yeast DNA from beer and other beverages Art. Nr.: FBD 0010; FBD 0100 Version 05/10 GEN-IAL GmbH Tel: +49 2241 2522980 Fax: +49 2241 2522989 info@gen-ial.de www.gen-ial.de - 1 -
First-Beer Magnetic DNA Kit Art. No. FBD 0010; FBD 0100 Version 05/10 1. Intended use Extraction of bacteria and yeast DNA from beer and other beverages with preenrichment. The DNA-extraction with the First-Beer Magnetic DNA Kit is a simple and rapid assay. Extraction of yeast and bacteria DNA from beer without preenrichment is also possible. Please use additional buffer set ZPS 0100. 2. Test principle The method is based on the biomagnetic separation of DNA. The isolated DNA is free of any PCR-inhibiting agents. 3. Package content 1x Beads suspension 1x Washing buffer 1x Enzyme solution All reagents should be stored at 2 8 C (35 46 F). 4. Reagents required but not provided 4.1 Equipment: heating block or water bath, minimum 65 C (140,8 F) microcentrifuge for 1.5-2 ml reaction tubes magnetic rack or magnetic particles transfer device (PickPen) Vortexer Pipettes: 10 200 µl, 100 1000 µl 4.2 Reagents and consumables sterile, double distilled or deionized water (ddh 2 O) sterile reaction tubes 1.5 ml sterile filter tips gloves for single use 100 % EtOH for yeast: Lyticase - 6 -
5. Warnings and precautions Avoid skin contact with the magnetic beads suspension. 6. Storage instructions Store all kit components at 2-8 C (35 46 F). During storage at 2 8 C (35 46 F) the magnetic particles sediment on the bottom of the flask. Shake the tube carefully until the particles are homogenously distributed. 7. Indications of deterioration of reagents Unknown. 8. Test implementation - Shake the magnetic beads suspension well before use - A preparation control to track potential carry-over or cross contaminations is highly recommended. - for yeast: prepare lyticase solution: 3 mg/ml lyticase buffer (= 50 % 1xTE+50 % Glycerol) 9. DNA-Extraction 9.1 bacteria 1. Transfer 1 ml of the pre-enriched bacteria culture in a 1.5 ml reaction tube and centrifuge at 14.000 rpm for 2 min. 2. Remove the supernatant carefully. No medium should remain within the reaction tube 3. Resuspend the pellet in 100 µl of the enzyme solution 4. Incubate this mixture for 10 min. at 65 C (140,8 F) 5. Add 400 µl homogenously mixed magnetic beads suspension 6. Incubate for 10 min. at 65 C (140,8 F) 7. Add 200 µl 100 % EtOH and vortex very well 8. Incubate this mixture for 2 min. at room temperature. Continue with step 10.1 / 10.2 9.2 yeast 1. Transfer 1 ml of the pre-enriched yeast culture in a 1.5 ml reaction tube and centrifuge at 14.000 rpm for 2 min. 2. Remove the supernatant carefully. No medium should remain within the reaction tube 3. Add 400 µl beads suspension and resuspend the bacteria pellet in this mixture 4. Add 14 µl lyticase 5. Incubate this mixture for 15-30 min. at 37 C (80 F) 6. Add 100 µl enzyme solution and incubate this mixture for 10 min. at 65 C (80 F) - 7 -
7. Add 200 µl 100% EtOH and vortex very well 8. Incubate this mixture for 2 min. at room temperature. Continue with step 10.1 / 10.2 10 Subsequent DNA-Isolation 10.1 Magnetic rack 10. Place the reaction tube in the magnetic rack. The magnetic particle move to the inner wall of the reaction tube due to the magnetic effect 11. Wait at least for 1 min. and remove the supernatant completely. No supernatant should remain within the reaction tube 12. Wash the DNA magnetic particle complex once with 800 µl of washing buffer, therefore take the reaction tube out of the magnetic rack and vortex the solution until it is completely mixed. Do not resuspend with the pipette 13. Place the tube in the magnetic rack and discard the supernatant after 1 min. 14. Take the reaction tube out of the magnetic rack, add 50 µl sterile dd H 2 O and resuspend the complex completely by pipetting 15. Incubate the reaction tube 10 min. at 65 C (80 F) 16. Place the reaction tube in the magnetic rack and wait 1 min., then transfer the DNA containing supernatant into a new tube 17. Use 1 5 µl of this DNA for PCR (10 µl may be optionally used, if necessary dilute DNA) 10.2 Magnetic Pen (PickPen) 10. Place each 1 x 800 µl washing buffer and 50 µl sterile dd H 2 O in different tubes 11. Use a PickPen with a new tip and fix the magnet and collect the magnetic particles of the lysate by dipping 12. Resuspend the magnetic particles completely in the washing buffer, for this unlock the magnet and mix the suspension well, e.g. by vortexing 13. Fix the magnet again and collect the magnetic particles out of the washing buffer by dipping 14. Resuspend the magnetic particles in 50 µl sterile dd H 2 O (unlock the magnet) 15. For elution of the DNA incubate the tube for 10 min. bei 65 C (80 F) 16. Fix the magnet and collect the magnetic particles and discard them 17. Use 1 5 µl of the DNA for PCR (10 µl may be optionally used, if necessary dilute DNA) - 8 -
11. List of abbreviations EtOH ethanol rpm rounds per minute (centrifuge Eppendorf 5415C) RT room temperature DNA desoxyribonucleic acid PCR polymerase chain reaction H 2 O water min minute or minutes GEN-IAL makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. If any materials are defective, GEN-IAL will provide a replacement product. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. GEN-IAL shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. - 9 -