Vacuum／SpinProtocolforIsolatingDNAfromBloodwithNucleatedRed

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

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. Insert the HiBind^? DNA column into the vacuum manifold. Carefully apply the 700ul lysate to an HiBind^? DNA column. Turn on the vacuum source to draw all liquid through the column. Turn off the vacuum.

Note: If the lysate has difficulty to pass through the column at this  stage. Place the column into a collection tube (supplied). Close the  lid and centrifuge at 10,000 x g for 5 minutes or until all liquid pass  through the column. Place the column into another collection tube  (supplied) and continue step 7 of the spin protocol.

8. Apply remaining lysate into the column. Turn on the vacuum source to draw all liquid through the column. Turn off the vacuum.

9. Add 500 ul of Buffer HB into the column. Turn on the vacuum source  to draw all liquid through the column. Turn off the vacuum.

10. Wash the column by pipetting 700 ul of DNA Wash Buffer diluted  with ethanol into the column. Turn on the vacuum source to draw all  liquid through the column. Turn off the vacuum.

11. Close the lid of HiBind^? DNA column, remove it from  the vacuum manifold. Insert the column into a collection tube (supplied)  and centrifuge at maximum speed for 2 minute to completely dry the  column.

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 tubes to sit for 5 min at room temperature.

13. To elute DNA from the column, centrifuge at maximum speed for 1  min. Retain flow-through containing the DNA. Place column into a second  1.5 ml tube. Elute DNA again as step 7-8. Discard column and store the  eluted DNA at -20°C.