Plus Blood Genomic DNA Purification Kit

Transcription

1 Plus Blood Genomic DNA Purification Kit Cat #:DP023P/ DP023P-150 Size:50/150 reactions Store at RT For research use only 1

2 Description: The Plus Blood Genomic DNA Purification Kit provides a rapid, simple and effective approach to isolate the genomic DNA from various animal bloods. The kit provides sufficient reagents to treat sample volume from 200 μl to 1.5 ml whole blood by three protocols. The process is based on a spin column format; the procedure involves cell lysis with proteinase K digestion, nucleic acids absorption and DNA elution. Typical yield ranges from 3-50 μg, depending on sample volume and species. DNA purified with this kit is suitable for various applications, including PCR, restriction enzyme digestion, cloning, dot blot analysis, etc. Components of the Kit: DP023P DP023P X RBC Lysis Solution 25 ml (dilute to 1X by 75 ml (dilute to 1X by water in final 250 ml) water in final 750 ml) 2. Binding/Extraction Solution* 30 ml 90 ml 3. Cell Lysis solution 25 ml 70 ml 4. Proteinase K Powder** 22 mg 22 mg x 3 5. RNase A Powder 22 mg 22 mg x 3 6. Wash Solution 16 ml (add 64 ml 48 ml (add 192 ml ethanol before use) ethanol before use) 7. Elution Solution 20 ml 60 ml 8. Spin Column 50 pcs 150 pcs 9. Collection Tube 50 pcs 150 pcs * Store the lyophilized Proteinase K & RNase A at -20 C, store all other kit components at room temperature(25~28 C). **Proteinase K may turn yellow after long term storage. This is a normal phenomenon. Materials to be supplied by the user: PBS buffer or TE buffer 100% Ethanol 2

3 Things to do before starting: Dissolve the Proteinase K Powder with 1.1 ml sterile H 2 O and store at -20 C. For purification for more than 200 μl whole blood (up to 1.5 ml), prepare 1X RBC Lysis solution in a new sterile bottle by adding sterile water to final 250 ml (DP023P) or 750 ml (DP023P-150). If RNA-free genomic DNA is required, dissolve the RNase A Powder with 220ul sterile H 2 O and store at -20 C. Turn on the water baths or heat blocks at 70 C, 60~65 C. Notes on blood sample: Blood sample should be stored at 4 C for no more than three days, or DNA may be degraded rapidly through apoptosis. If blood sample is stored at freezer, thaw frozen blood quickly at 37 C and extraction immediately. When treating with nucleated blood (e.g. fish, frog or bird), use only protocol A and sample volume should be no more than 20 μl. General Procedure: Sample preparation 1. A. Whole blood 200 μl a) In 2 ml centrifuge tube, add 3X volumes of 1X RBC Lysis Solution to whole blood, mix by vortex. (if sample less than 200 μl, add PBS or TE buffer to final 200 μl.) b) Incubate at room temperature for 5 min or longer until red blood cell completely lysis (solution become clear red). c) Centrifuge at 3,500 x g for 10 min and remove the supernatant. d) If RNA-free DNA is desired, add 4 μl RNaseA solution and incubate at room temperature for 5 min. e) Add 20 μl Proteinase K Solution, mix by vortex. Proceed to step 2. 3

4 B. Whole blood 200~400 μl a) In 2 ml centrifuge tube, add 3X volumes of 1X RBC Lysis Solution to whole blood, mix by vortex. b) Incubate at room temperature for 5 min or longer until red blood cell completely lysis (solution become clear red). c) Centrifuge at 3,500 x g for 10 min and remove the supernatant. d) Resuspend the pellet with 200 μl PBS or TE buffer. e) If RNA-free DNA is desired, add 4 μl RNaseA solution and incubate at room temperature for 5 min. f) Add 20 μl Proteinase K Solution, mix by vortex. Proceed to step 2. C. Buffy Coat (from whole blood 1. 5 ml) a) Centrifuge whole blood at 3,300 x g for 10 min using swing bucket rotor. Three fractions are form: The upper clear layer is plasma, the interface (middle layer) is buffy coat (leukocyte-concentrated fraction), and the bottom layer contains concentrated erythrocytes. b) Carefully pipet the interface with a clean dropper into a new 15 ml centrifuge tube. c) Add 3X volumes of 1X RBC Lysis Solution to buffy coat and vortex to mix. d) Incubate at room temperature for 5 min or longer until red blood cells completely lysis (solution become clear red). e) Centrifuge at 3,500 x g for 5 min and remove the supernatant. f) Resuspend the pellet with 200 μl of PBS or TE buffer. g) If RNA-free DNA is desired, add 4 μl RNaseA and incubate at room temperature for 5 min. h) Add 20 μl Proteinase K Solution, mix by vortex. Proceed to step Add 400ul Cell Lysis Solution, mix by pulse vortex 15~30 sec. 3. Add 500 μl Binding/Extraction Solution, mix by pulse vortex 15~30 sec. * It is normal that white precipitate appears after adding Binding/Extraction Solution. 4

5 4. Incubate at 70 C for 10 min with occasional pulse vortexes for 5~10 sec during incubation. 5. Centrifuge at top speed (12,000~14,000 x g) for 5 min to remove the debris, which may clog the column. 6. Collect the supernatant into a new centrifuge tube without disturbing the pellet. 7. Add 100% Ethanol to the supernatant. Please refer to the table as below. Fresh whole blood 100% Ethanol 200 μl 200 μl >200 μl 500 μl Mix thoroughly by vortexing and apply the solution to spin column with collection tube and centrifuge at top speed for 1 min. * A precipitate may form after addition of ethanol, apply all the solution and precipitate to the column with collection tube. 8. Discard the filtrate, add 700 µl of Wash Solution and centrifuge at top speed for 1 min. Repeat this step once more. 9. Discard the filtrate and centrifuge for additional 5 min at top speed to remove residual trace of ethanol. * If centrifugation speed is lower than 12,000x g or residual ethanol must be removed completely, incubate the spin column in a heat oven (60~65 C) for 5 min to evaporate all of the ethanol. 10. Transfer the spin column into a new microcentrifuge tube and add 100~200 μl preheated (60~65 C) Elution Solution or H 2 O (ph 7.0~8.5) into the column and wait for 1~2 min. 11. Centrifuge at top speed for 1 min to elute the DNA. Store the eluted DNA at -20 C. * Repeat elution once will have 10~15% more DNA yield, but DNA concentration will be diluted. 5

6 Troubleshooting Guide Clogging of Spin Column a) Sample volume exceeded the recommended volume Comments and Suggestion If using more than 1.5 ml whole blood sample, separate into multiple tubes. b) Sample tissue was not lysed completely. c) Sample contained clots Brownish residues persist on the membrane of Spin Column after washing a) Incomplete digestion of hemoglobin b) No alcohol added to the supernatant (Step 7) before loading onto the Spin Column. c) Incorrect amount of ethanol added to the Wash Solution. Poor DNA yield a) Sample may be old or degraded. 1) Add additional Proteinase K in Step1 and extend the incubation time in Step 4. 2) Do not use a combined solution (Proteinase K + Extraction Solution) that has been stored. Do not use blood clot to purify DNA. Use whole blood sample mixed well with anticoagulant to prevent clotting. Vortex the sample after Proteinase K is added. Mix the sample every 3~5 minutes during incubation. Before loading onto Spin Column, add recommended volume (more than 200 μl or suitable volume) of absolute ethanol or isopropanol to the sample. Make sure that recommended volume of ethanol was added into the Wash Solution (component 6) bottle during initial preparation of Wash Solution. The yield will vary among individual samples of fresh or aged (older than 24 hours) whole blood. For optimum yield, use whole blood within a few hours after collection. b) Wash Solution was Make sure that recommended volume of 6

7 not prepared as recommended. c) Sample was not lysed completely d) DNA was not eluted properly. Poor performance in downstream enzymatic applications. a) Residual ethanol contamination b) Genomic DNA was degraded Poor quality of Genomic DNA(A260/A280 ratio) a) Genomic DNA is contaminated with ethanol was added into the Wash Solution (component 6) bottle during initial preparation of Wash Solution. 1) Add additional Proteinase K in the Step 1 and extend the incubation time in the Step 4. 2) Do not use a combined solution (Proteinase K + Extraction Solution) that has been stored. 1) DNA should be eluted with a low-salt solution [e.g., Elution Solution (10 mm Tris Cl, ph 8.5) or water] Elution efficiency is dependent on ph. The maximum efficiency is achieved between ph 7.0 and 8.5. When using water for elution, make sure that the ph of water is within this range. 2) Ensure that Elution Solution is added onto the center of the membrane and is completely absorbed. 3) Allow the Elution Solution to incubate in the column for longer time. 4) Elute twice to increase the DNA recovery. Following the Wash Step, dry the Column with additional centrifugation at 12~14,000 x g for 5 min or incubate at 60~65 C for 5 min. Use fresh samples or freeze fresh samples in liquid nitrogen immediately and store at -80 C. Add RNase A Solution to the sample as described in the Step 1. 7

8 RNA b) Eluted genomic DNA contains contaminants. Do not touch the rim of the column during sample or buffer loading. 8

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