ProtocolforIsolatingDNAfromBloodwithNucleatedRedbloodCells

實驗概要

DNA  isolation from fish or avian blood sample can be difficult because it  contains nucleated red blood cells. E.Z.N.A. NRBC Blood DNA Kit is  designed for isolating genomic DNA from fresh, or ethanol preserved  blood samples containing nucleated red blood cells. The kit allows  single or multiple, simultaneous processing of samples in under 60  minutes. There is no need for phenol/chloroform extractions, and  timeconsuming steps such as CsCl gradient ultracentrifugation are  eliminated. Purified DNA obtained with the E.Z.N.A.^? Blood DNA Kit will be ready for applications such as PCR, Southern Blotting, and Restriction Digestion.

E.Z.N.A.^? NRBC Blood DNA Kit uses the reversible nucleic acid-binding properties of HiBind^?  matrix, combined with the speed of mini-column spin technology. A  specifically formulated buffer system allows genomic DNA 30-60 kb to  bind to the matrix. Samples are first lysed under denaturing conditions  and then applied to the HiBind^? DNA spin columns to which DNA  binds, while cellular debris, hemoglobin, and other proteins are  effectively washed away. High quality DNA is finally eluted in sterile  deionized water or low salt buffer.

主要試劑

1. Ethanol - approximately 0.3 ml per sample.

2. RNase A - Prepare a stock solution of RNase A at 25 mg/ml.

主要設備

1. Tabletop microcentrifuge and sterile 1.5 ml tubes.

2. Water bath - set to 65°C.

實驗步驟

NOTE: The procedure below has been optimized for use with FRESH or ethanol fixed blood samples of 1 to 15 ul in volume.

Bring samples and Proteinase K solution to room temperature and have a  water bath equilibrated to 65°C. Preheat an aliquot of Elution Buffer  (approximately 0.4 ml per sample) at 65°C. Carry out all centrifugation  steps at room temperature.

1. Add 1 - 4 ul of blood sample to a sterile microcentrifuge tube.  Add 250 ul of RL1 Buffer. Mix throughly by vortexing for 10 seconds.

2. Add 25 ul Proteinase K (20mg/ml) and 275 ul of Buffer BL. Vortex at maxi speed for 15s to mix thoroughly.

3. Incubate the sample at 65°C for 10 min.

4. Briefly vortex the tube once during incubation. Optional: If  RNA-free genomic DNA is required, add 5ul RNase A (25mg/ml) to each  sample and incubate at room temperature for 5 minutes .

5. Add 275 ul of absolute ethanol (room temperature, 96-100%) to  lysate and vortex at maxi speed for 20s to mix thoroughly. Briefly  centrifuge the tube to collect any drops from the inside of the lid.

6. Assemble an HiBind^? DNA column in a 2 ml collection  tube (provided). Add 100ul of Equitation Buffer to each column. Wait 3-4  minutes at room temperature. Centrifuge at maximum speed for 30  seconds.

7. Transfer the lysate from step 5 into the column and centrifuge at  $10,000 x g for 1 min to bind DNA. Discard the collection tube and  flow-through liquid.

8. Place the column into a second 2 ml tube (provided) and add 500 ul  of Buffer HB. Centrifuge at $10,000 x g for 1 min. Discard flow-through  liquid and reuse the collection tube for next step.

9. Place the column into a same 2 ml tube from step 7 and wash the  column by pipetting 700 ul of DNA Wash Buffer diluted with ethanol.  Centrifuge at $10,000 x g for 1 min. Again, dispose of collection tube  and flow-through liquid.

Note: DNA Wash Buffer is provided as a concentrate and must be  diluted with absolute ethanol as indicated on the bottle and page 3. If  refrigerated, the diluted DNA wash buffer must be brought to room  temperature before use.

10. Using a new collection tube, wash the column with a second 700 ul  of DNA Wash Buffer and centrifuge as above. Discard flow-through and  re-use the collection tube for next step.

11. Place the empty column into the same 2 ml collection tube form  step 10, centrifuge at maximum speed for 2 min to dry the column. This  step is crucial for ensuring optimal elution in the following step.

12. Place the column into a sterile 1.5 ml microfuge tube and add  100-200 ul of preheated (65°C) Elution Buffer (10mM-Tris-HCl, pH 8.5).  Allow o tubes to sit for 5 min at room temperature.

13. To elute DNA from the column, centrifuge at maximum speed  ($14,000 x g) for 1 min. Retain flow-through containing the DNA. Place  column into a second 1.5 ml tube. Elute DNA again as step 11-12. Discard  column and store the eluted DNA at -20°C.

Note: First elution typically yields 60%-70% of the DNA bound to the  column. Thus two elution generally give >90%. However, increasing  elution volume reduces the concentration of the final product. To obtain  DNA at higher concentrations, elution can be carried out using 50 ul to  100 ul Elution Buffer. Volumes lower than 50 ul greatly reduce yields.  Alternatively use the first eluate to perform the second elution.

If necessary the DNA can be concentrated. Add sodium chloride to a  final concentration of 0.1 M followed by 2 x volume of absolute (100%)  ethanol. Mix well and incubate at -20°C for 10 min. Centrifuge at 14,000  x g for 10 min and discard supernatant. Add 700 ul of 80% ethanol and  centrifuge at 10,000 x g for 2 min. Discard supernatant, air dry the  pellet (2 min) and resuspend DNA in 20 ul sterile deionized water or 10  mM Tris-HCl, pH 8.