TIANamp Blood DNA Midi Kit

Transcription

1 TIANamp Blood DNA Midi Kit For purification of high-purity genomic DNA from ml blood samples

2 DP TIANamp Blood DNA Midi Kit Kit Contents (Spin Column) Cat. no. DP Contents Buffer GE Buffer GD Buffer PW Buffer TB Proteinase K 10 preps 40 ml 13 ml 15 ml 15 ml 2 x 1 ml Spin Columns CB5 10 Collection Tubes 15 ml 20 Compatible Reagents Handbook 1 RNase A (100 mg/ml, Cat. no. RT405-12); Liquefaction Columns CX2 (Cat. no. RK166) Storage TIANamp Blood DNA Midi Kit could be stored at dry and room temperature (15-25 C) for up to 12 months without any reduction in performance and quality. For long-term storage, 2-8 C is recommended. Introduction TIANamp Blood DNA Midi Kit is specially designed for purification of genomic DNA from ml fresh or frozen stored whole blood (Samples collected in blood collection tubes treated with EDTA, TIANamp Blood DNA Midi Kit Handbook 1

3 heparin or citrate), clotted blood and white blood cells. It adopts innovative silica-based material and special buffer system for effective binding and extracting gdna from blood samples. The procedure completely removes contaminants and enzyme inhibitors, making total DNA isolation fast, convenient and reliable. Genomic DNA isolated with this kit is of high-quality and serves as an excellent template for agarose gel analysis, restriction enzyme digestion, PCR analysis and blotting procedures. DNA Yield: μg gdna from 1 ml mammalian blood (treated with anticoagulant) Important Notes 1. Repeated freezing and thawing of stored samples should be avoided, since this will lead to DNA size and volume reduction. 2. If precipitates formed in Buffer GE, dissolve them by incubating at 37 C. 3. Do use a bench centrifuge for all centrifugation steps of TIANamp Blood DNA Midi Kit. All centrifugation steps are performed at room temperature (15-25 C). 4. The 15 ml centrifuge tubes (Cat. no ) used in centrifugation of step 1-5 should be self-provided (Make sure the Spin Column and the centrifuge tube fit for each other before centrifugation). 5. For clotted blood templates, liquefaction Column is recommended and should be self-provided (Cat. no. RK166). Protocol Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly. 1. Dispense 200 μl Proteinase K (20 mg/ml) into the bottom of a 15 ml centrifuge tube. TIANamp Blood DNA Midi Kit Handbook 2

4 2. Preparation of samples. a. For blood samples, pipet ml blood and mix briefly. b. For clotted blood samples, firstly put a blood clot Liquefaction Column CX2 (not provided, Cat. no. RK166) into a 15 ml centrifuge tube prepared in step 1, then add ml clotted blood into CX2, centrifuge at 4,600x g (~6,000 rpm) for 1 min. Note: Blood samples and clotted blood samples after treatment could be pipetted into centrifuge tube directly, then add Proteinase K and mix thoroughly. 3. Add 2.4 ml Buffer GE, and mix thoroughly by vigorous shaking for 30 sec. Note: For samples up to 2-3 ml, Buffer GE could be increased to 3.6 ml. For RNA cleaning up, 40 μl RNaseA solution (100 mg/ml, not provided, Cat. no. RT ) should be added into the centrifuge tube, mix thoroughly by vigorous shaking for 15 s, then stand at room temperature for 5 minutes. 4. Incubate at 65 C for 10 min, mix thoroughly by vigorous shaking every 3 minutes. Centrifuge briefly to collect residual liquid from the sides of the tube (Longer incubation time will be helpful for adequate lysis for special samples). 5. Add 2 ml ethanol (96-100%) to the sample, and mix by vortex. A white precipitate may form on addition of ethanol. Note: Cold down the sample to room temperature before the ethanol (96-100%) adding. For 2-3 ml blood samples, 3 ml ethanol (96-100%) is recommended. 6. Pipet half of the mixture from step 4 into the Spin Column CB5 (in a 15 ml collection tube) and centrifuge at 1,850x g (~3,000 rpm) for 3 min. Discard flow-through and place the spin column into the collection tube. 7. Repeat step 6 with the other half of the mixture from step 4 in the same Spin Column CB5. TIANamp Blood DNA Midi Kit Handbook 3

5 8. Add 2 ml Buffer GD (ensure ethanol has been added) to Spin Column CB5, and centrifuge at 4,500x g (~5,000 rpm) for 1 min, then discard the flow-through and place the spin column into the collection tube. 9. Add 2 ml Buffer PW (ensure ethanol has been added) to Spin Column CB5, and centrifuge at 4,500x g (~5,000 rpm) for 1 min. Discard the flow-through and place the spin column into the collection tube. 10. Add 2 ml Buffer PW (ensure ethanol has been added) to Spin Column CB5, and centrifuge at 4,500x g (~5,000 rpm) for 15 min. Discard the flow-through and place the spin column into the a new 15 ml centrifuge tube. Note: The long-term centrifugation dries the spin column membrane, ensuring that no ethanol is carried during DNA elution. Residual ethanol may interfere with down-stream reactions. 11. Pipet 300 μl Buffer TB directly to the center of the membrane. Incubate at room temperature (15-25 C) for 5 min, and then centrifuge at 4,500x g (~5,000 rpm) for 2 min. Note: The volume of elution buffer should not be less than 200 μl, or it may affect the recovery efficiency. For high yield of DNA, adding the flow-through to the center of membrane, incubating at room temperature (15-25 C) for 2 min and centrifuging at 4,500x g (~5,000 rpm) is recommended. ph value of elution buffer has great influence on elution recovery, we suggest using buffer TB (ph>7.0) to elute gdna. For long-term storage, DNA should be stored at -20 C is to avoid acid hydrolysis of DNA. TIANamp Blood DNA Midi Kit Handbook 4

6 Ordering Information DNA Purification Product Size Cat. no. TIANamp Blood DNA Kit (0.1-1 ml) 50 preps 200 preps DP DP DNA Marker Product Size Cat. no. λ DNA/Hind III 50 preps 200 preps MD MD PCR MasterMix Product Size Cat. no. 2 Taq PCR MasterMix (with loading dye) 1 ml 5 1 ml KT KT Taq Plus PCR MasterMix (with loading dye) 0.5 ml 5 1 ml KT KT TIANamp Blood DNA Midi Kit Handbook 5