Protocol for Isolating DNA from Blood with Nucleated Red blood Cells
实验概要
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.
主要试剂
1. Ethanol - approximately 0.3 ml per sample.
2. RNase A - Prepare a stock solution of RNase A at 25 mg/ml.
主要设备
1. Tabletop microcentrifuge and sterile 1.5 ml tubes.
2. Water bath - set to 65°C.
实验步骤
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.
