Standard Protocol For Up to 10 ml Whole Blood
实验概要
The E.Z.N.A.® Blood DNA Maxi Kit is designed for isolation of genomic DNA from up to 25 ml of fresh, whole blood treated with any common anticoagulant such as heparin, EDTA, or acid-citrate-dextrose. 20ml of blood typically yields 700–1000 ug of genomic DNA. The procedure completely removes contaminants and enzyme inhibitors making total DNA isolation fast, convenient, and reliable. There is no need for phenol/chloroform extractions, and time-consuming steps such as CsCl gradient ultracentrifugation, and precipitation with isopropanol or LiCl, are eliminated. DNA purified using the E.Z.N.A.® Blood DNA method is ready for applications such as PCR*, Restriction digestion, Southern blot and so on.
The E.Z.N.A.® Blood DNA Maxi Kit uses the reversible binding properties of HiBind® matrix, a new silica-based material. This is combined with the speed of maxi-column spin technology. Red blood cells are selectively lysed and white cells collected by centrifugation. After lysis of white blood cells under denaturing conditions that inactivate DNases, genomic DNA is purified on the HiBind® Maxi spin column. A specifically formulated high salt buffer system allows DNA molecules greater than 200 bases to bind to the matrix. Cellular debris and other contaminants (such as hemoglobin) are effectively washed away and high quality DNA is finally eluted in Elution Buffer.
This protocol allows rapid isolation of genomic DNA from up to 10 ml blood sample. Yield vary depend on source. Do not use more than 2 x 108 cells
主要试剂
1. Absolute ethanol - approximately 3 ml per sample
主要设备
1. Water bath - set to 70°C.
2. Have a shaking water bath set to 55°C.
3. 50 ml centrifuge tubes capable of 4000 x g
4. Laboratory centrifuge equipped with swinging-bucket rotor.
实验步骤
1. Add up to 10 ml whole blood to a 50 ml centrifuge tube. If the sample less than 10 ml, bring the volume up to 10 ml with 10 mM Tris-HCl, PBS, or Elution Buffer provided.
2. Add 250 ul OB Protease, 10.2 ml Buffer BL and 20 ul RNase A. Vortex at max speed for 5 minutes to mix thoroughly.
3. Incubate sample at 70°C for 10 min. Briefly vortex the tube once during incubation.
4. Add 10.3 ml absolute ethanol (room temperature, 96-100%) to lysate and mix throughly by vortexing at max speed for 30s.
5. Insert a HiBind® DNA Maxi column in a 50 ml collection tube (provided). Transfer 20 ml of the lysate from Step 4 into the column and centrifuge at 4,000 x g for 5 min to bind DNA. Discard the flow-through liquid and re-use the collection tube.
NOTE: Since the HiBind® DNA Maxi column can only contains around 20 ml sample volume, it is necessary to load the column twice.
6. Place the column back into the 50 ml collection tube and load the remaining of the lysate for step 4 into the column. Centrifuge as above. Discard the flowthrough and re-use the collection tube.
7. Place the column into the same 50 ml tube and wash by pipetting 5 ml of HB Buffer. Centrifuge at 4,000 x g for 5 minutes Discard the flow-through liquid and re-use the collection tube.
8. Place the column into the same 50 ml tube from step 7 and wash by pipetting 10 ml of DNA Wash Buffer diluted with ethanol. Centrifuge at 4,000 x g for 5 minutes. Discard the collection tube and flow-through liquid.
9. Using a new 50 ml centrifuge tube, wash the column with a second 10 ml of Wash Buffer diluted with ethanol and centrifuge as above. Discard flowthrough.
10. Using the same 50 ml collection tube, centrifuge at 4000 x g for 10 min to dry the column. This step is critical for removal of trace ethanol that might otherwise interfere with downstream applications.
11. Place the column into a nuclease-free 50 ml centrifuge tube and add 1 ml of preheated (70°C) Elution Buffer. Allow tubes to sit for 5 min at room temperature.
12. To elute DNA from the column, centrifuge at 4,000 x g for 5 min. Retain flow-through containing the DNA. Place column into a second 50 ml tube and repeat elution step 11-12 with another 1 ml of preheated Elution Buffer. Discard column.
Note: Each elution typically yields 60%-70% of the DNA bound to the column. Thus two elutions generally give >80%. However, increasing elution volume reduces the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 0.5 ml Elution Buffer. Volumes lower than 500 ul greatly reduce yields. Alternatively, use the first eluate to perform the second elution
