Simply Blood DNA Mini Kit Manual SG-P-023-1
Transcription
Simply Blood DNA Mini Kit Manual SG-P-023-1
Simply Blood DNA Mini Kit Manual SG-P-023-1 Contents Contents 1 General Information Introduction . . . . . . . . Intended use . . . . . . . Product specifications . . . List of Components . . . . Storage conditions . . . . Safety information . . . . . Hazard Identification 1 . . . . . . . 2 2 2 2 3 3 3 4 2 Additional Requirements Required equipment and additives . . . . . . . . . . . . . . . . . . . . . . . . . Required components not included . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 5 3 Protocol Recommended steps before use . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6 6 4 Troubleshooting 8 5 Warranties and Quality Control 9 6 Technical support Legal Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 10 7 Your notes 11 Simply Blood DNA Mini Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 1 1 General Information Introduction The Simply Blood DNA Mini Kit provides a very fast and simple method for the preparation of total DNA from whole blood samples. The extraction procedure is based on binding nucleic acids to the surface of a Spin Filter membrane and consists of four steps. During the first step, cells and nuclei are lysed. Subsequently, the DNA is bound to a Spin Filter membrane, washed and, finally, eluted. Intended use The kit is designed for the preparation of DNA from whole blood samples. It is suitable to process up to 400 µl of fresh or frozen whole blood, stabilized with EDTA or citrate. The extracted DNA can be used for a wide range of different molecular biology downstream applications, including PCR, restriction digestion, cloning and sequencing. B For Research Use Only — Not for use in diagnostic procedures. Product specifications Starting Material Fresh or frozen whole blood (200 or 400 µl), stabilized with EDTA or citrate Time for preparation approx. 30 minutes Binding capacity more than 60 µg genomic DNA Expected Yield at least 30 µg genomic DNA, depending on initial sample volume Purity indicator Ratio A260 :A280 between 1.7 and 2.0 Order number SG-P-023-1 (50 reactions) Manual version SG-P-023-1-1 Page 2 Simply Blood DNA Mini Kit List of Components Component Quantity Storage Cat No Binding Solution C 40 ml 15 - 25°C SG-C-031-1 Collection Tube B 250 pcs 15 - 25°C SG-C-009-1 Elution Buffer A 6x 2 ml 15 - 25°C SG-C-063-1 Elution Tube A 50 pcs 15 - 25°C SG-C-038-1 Lysis Solution D 25 ml 15 - 25°C SG-C-055-1 Proteinase K II 2 pcs 4°C (if dissolved: -20°C) SG-C-022-1 Spin Filter Red A 50 pcs 15 - 25°C SG-C-018-1 Washing Solution A 25 ml 15 - 25°C SG-C-044-1 Washing Solution G 8 ml 15 - 25°C SG-C-045-1 Storage conditions All components of the Simply Blood DNA Mini Kit except for the Proteinase K II should be stored dry at room temperature (15 to 25°C). Lyophilized Proteinase K should be stored at 4°C. Dissolved Proteinase K should be stored at -20°C. Repeated freezing and thawing should be avoided. Allocation of the solution into aliquots is recommended. If there are any precipitates within the provided solutions solve these precipitates by careful warming. Do not use the kit after the expiration date. Safety information All reagents in this kit have to be used for the intended use described above only. The kit should be used by authorized staff only. It is strongly recommended to follow the instruction for use. Inappropriate handling or use of the kit or non-observance of the instructions for use can increase the danger for your health and reduce the reliability of results. The reagents should be stored inside the original vessels at the given storage temperatures as indicated on the Fact Sheet. Single components of different lots and consumables should not be exchanged. The expiry dates have to be considered. Please pay attention to the regulations for the prevention of accidents in laboratories. In particular, the following safety precautions have to be considered: B Don’t eat, drink or smoke! Always wear protective clothing, safety glasses and gloves! Do not add bleach or acidic components to the waste after sample preparation! All liquid waste must be considered as potentially infectious and must be handled and discarded according to local safety regulation! Simply Blood DNA Mini Kit Page 3 For more information, please read the Fact Sheet and ask for the material safety data sheets (MSDS’s). Emergency medical information in English and German can be obtained 24 hours a day from the Poison Information Center in Freiburg (Germany), Phone: +49 761 19240. Hazard Identification Please read carefully the following hazard identification information that classifies the substances or mixtures contained in this product. Proteinase K II Signal word DANGER Pictograms Hazard statement(s) H315 Causes skin irritation. H319 Causes serious eye irritation. H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled. H335 May cause respiratory irritation. Precautionary statement(s) P261 Avoid breathing dust/fume/gas/mist/vapours/spray. P305 + P351 + P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P342 + P311 If experiencing respiratory symptoms: Call a POISON CENTER / doctor / ... R-phrase(s) R32 Contact with acids liberates very toxic gas. R36/37/38 Irritating to eyes, respiratory system and skin. S-phrase(s) S22 Do not breathe dust. S24 Avoid contact with the skin. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S36/37 Wear suitable protective clothing and gloves. Page 4 Simply Blood DNA Mini Kit 2 Additional Requirements Required equipment and additives 2 Microcentrifuge 2 Thermomixer 2 Vortexer 2 Variable pipettes for 10 µl – 1 000 µl 2 Sterile pipette tips with protection against contamination Required components not included 2 1.5 ml reaction tubes 2 optionally RNase A (100 mg/ml) 2 96 - 99.8 % ethanol 2 ddH2 O Simply Blood DNA Mini Kit Page 5 3 Protocol This protocol is valid for the preparation of total DNA of 200 or 400 µl whole blood (fresh or frozen, stabilized with EDTA or citrate). Recommended steps before use Before first use: 2 Washing Solution G: Add 72 ml of ethanol (96 – 99.8 %) 2 Proteinase K II: Add 1.5 ml of ddH2 O, mix thoroughly and store in aliquots at -20°C Before use: • Thermomixer: Preheat to 60°C • Elution Buffer A: Preheat to 60°C • Make sure that all other components have room temperature Procedure 1. Add 200 / 400 µl of blood into a 1.5 ml reaction tube. Note: If the blood volume is less than 200 / 400 µl, add the appropriate volume of PBS. 2. Add 200 / 400 µl of Lysis Solution D and 20 / 30 µl Proteinase K II to the sample. Mix by pulsed vortexing for 10 seconds. 3. Incubate at 60°C for 10 minutes at 550 rpm. Note: If required, add 4 µl of an RNase A stock solution (100 mg/ml) to remove RNA from sample, vortex shortly and incubate for 5 minutes at room temperature. 4. Add 350 / 700 µl of Binding Solution C to the lysed sample. Mix carefully by pipetting up and down 3 – 4 times. Don’t vortex! 5. Place the Spin Filter Red A into a Collection Tube B. Page 6 Simply Blood DNA Mini Kit 6. Apply the sample to the Spin Filter Red A and close the cap. Centrifuge at 11 000 x g for 1 minute. Attention: The 400 µl blood - protocol needs to divide the sample into two parts. First add 750 µl of the sample to the Spin Filter Red A and close the cap. Centrifuge at 11 000 x g for 1 minute. Place the Spin Filter Red A into a new Collection Tube B and discard the used Collection Tube B. Add the residual sample to the Spin Filter Red A. Close the cap and centrifuge at 11 000 x g for 1 minute. 7. Place the Spin Filter Red A into a new Collection Tube B and discard the used Collection Tube B. 8. Add 400 µl of Washing Solution A and close the cap. Centrifuge at 11 000 x g for 1 minute. 9. Place the Spin Filter Red A into a new Collection Tube B and discard the used Collection Tube B. 10. Add 600 µl of Washing Solution G and close the cap. Centrifuge at 11 000 x g for 1 minute. 11. Place the Spin Filter Red A into a new Collection Tube B and discard the used Collection Tube B. 12. Add 600 µl of Washing Solution G and close the cap. Centrifuge at 11 000 x g for 1 minute. 13. Place the Spin Filter Red A into a new Collection Tube B and discard the used Collection Tube B. 14. Centrifuge at max. speed for 3 minutes (above 11 000 x g) to avoid a carry-over of ethanol residuals. 15. Place the Spin Filter Red A into the 1.5 ml Elution Tube A. 16. Add 200 µl of Elution Buffer A (preheated to 60°C) and incubate at room temperature for 2 minutes. Centrifuge at 11 000 x g for 1 minute. Note: To increase the concentration of the extracted DNA, elute in two steps with preheated Elution Buffer A (e.g. 100 µl + 100 µl) or elute with volumes smaller than 200 µl. 17. Store the extracted DNA at 4°C. Long term storage of DNA should happen at -20°C. Simply Blood DNA Mini Kit Page 7 4 Troubleshooting Problem Probable Cause Remedy Increase lysis time Slow flow speed through the Spin Filter Red A Insufficient lysis Overloading of Spin Filter reduces yields Increase centrifugation speed / time Insufficient lysis See above Blood sample may contain too few white blood cells Draw new blood sample Blood sample too old Storage at 2 to 5°C for more than 5 days may reduce the yields Insufficient mixing with Binding Solution C Mix carefully by pipetting up and down 3 – 4 times. Don’t vortex! Poor DNA yield Incomplete elution Increase the incubation time with Elution Buffer A to 5 minutes Repeat Elution step once again Increase the Elution Buffer A volume Too much Elution Buffer A Elute the DNA with lower Elution Buffer A volume Incorrect storage of the sample Avoid thawing and freezing Degraded or sheared DNA Low performance of downstream applications RNA contamination Page 8 Improper collection of the blood sample Draw new blood sample Incorrect storage of the DNA Avoid thawing and freezing Salt carry-over Ensure proper washing Check Wash Buffers for salt precipitates Ethanol carry-over Increase centrifugation time for ethanol removal RNA Digest RNA with RNase Simply Blood DNA Mini Kit 5 Warranties and Quality Control Simply Genomics guarantees the correct performance of each kit for applications as described in the instruction for use. The purchaser must determine the suitability of the product for its particular use. We reserve the right to change, alter or modify any product to enhance its performance and design. If a Simply Genomics product does not meet your expectations or if you are unsatisfied for any reason with this product, please contact our Technical Assistance. Simply Blood DNA Mini Kit Page 9 6 Technical support If you have any questions regarding any aspects of this product, please do not hesitate to contact us via [email protected] or call us at +49 (0) 3641 / 777 888. Legal Information This product has been manufactured, distributed and published by: Moldiax GmbH Konrad-Zuse-Str. 1 07745 Jena Germany Phone: +49 (0) 3641 / 777 333 E-Mail: [email protected] Internet: www.moldiax.de © Copyright 2014, Moldiax GmbH Page 10 Simply Blood DNA Mini Kit 7 Your notes Simply Blood DNA Mini Kit Page 11 Page 12 Simply Blood DNA Mini Kit Moldiax GmbH Konrad-Zuse-Straße 1 07745 Jena / Germany Telefon +49 (0) 36 41 / 777 333 Telefax +49 (0) 36 41 / 777 67 333 [email protected] www.simplygenomics.com