Maxi-Yield protocol for 10- 20 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 Modified protocol allows isolation of genomic DNA from up to 20  ml blood sample. Yield vary depend on source. Using more than 25 ml  blood is not recommend.

主要试剂

Red Blood Lysis Buffer (ERL Buffer):

NH₄Cl 155mM

KHCO₃ 10mM

Na₂EDTA 0.1mM

Adjust to pH 7.4 with 1M Hcl or NaOH

实验步骤

1. Divide blood  sample into two 50 ml tube with equal volume. To 1 volume of whole fresh  blood add 5 volumes of 1 x Red Blood Lysis Buffer (ERL Buffer. For  example, add 50 ml Buffer ERL to 10 ml blood. Mix by vortexing).

2. Incubate for 15 min on ice, mixing by brief vortexing twice. Lysis  of red blood cells is indicated when the solution becomes translucent.  Blood samples from individuals with an elevated hematocrit or ECR,  extend incubation time to 20 min.

3. Pellet leukocytes by centrifuging at 450 x g for 10 min at 4°C.  Completely remove and discard the supernatant containing lysed red blood  cells.

4. Wash the white blood cell pellet with 2 volumes of ERL Buffer per  volume of whole blood used in step 1. Thoroughly vortex to resuspend  cells.

Tip: If you used 10 ml of whole blood, wash with 20 ml of Buffer ERL.

5. Centrifuge at 450 x g for 10 min at 4°C. Again, completely remove and discard the supernatant.

6. Resuspend the cell pellet with 0.5 ml PBS Buffer and 3ml TL buffer  to the pelleted white blood cells in each tube and vortex thoroughly to  mix. Combine the sample into one 50 ml tube, there should be 7ml total  sample after the combination.

7. Add 250ul of OB protease and vertex to mix well. Incubate at 55°C  in a shaking water bath to effect complete lysis. If no shaking water  bath is available, vortex every 20-30 minutes. Lysis time depend on  amount and type of tissue, but usually under 2 hours.

8. Add 20 ul RNase A, 7 ml Buffer BL and mix thoroughly by vortexing  at max speed for 30s. Incubate the mixture at 70oC for 10 minutes. A  wispy precipitate may form on addition of Buffer BL, but does not  interfere with DNA recovery.

9. Add 7 ml absolute ethanol and mix thoroughly by vortexing at max  speed for 30s. If precipitation can be seen at this point, break the  precipitation by passing through a needle using a syringe.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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