StandardProtocolForUpto10mlWholeBlood

實驗概要

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

主要試劑

1. Absolute ethanol - approximately 3 ml per sample

主要設備

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.

實驗步驟

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