IsolateDNAfromNRBCBloodfromBuccalSwabcollectedwithfilterpaper

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. Absolute Ethanol - approximately 0.3 ml per sample

2. RNase A(Optional) - Prepare a stock of RNase A at 50mg/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. Place the  buccal swab or paper contains dried blood spot in a 2 ml centrifuge tube  and add 500 ul RS1 Buffer in the tube. If RNA-free DNA is required for  downstream application, add 4 ul RNase A (25mg/ml) into the sample.

2. Add 25ul OB Protease and 500ul Buffer BL into sample. Mix by vortexing at maxi speed for 30s.

3. Incubate at 65°C for 10 minutes. Collect any liquid drop from lid by brief centrifugation.

4. Centrifuge at 14,000 x g for 10 minutes. Carefully transfer the supernatant to a new 1.5 ml tube.

5. Add equal volume of absolute ethanol (room temperature, 96-100%)  to the sample and mix by vortexing at maxi speed for 15 seconds. Collect  any liquid drop from lid by brief centrifugation.

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. Carefully apply entire sample into HiBind^? DNA. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and collection tube.

8. Place the column into a new 2 ml collection tube (provided).  Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and  collection tube.

9. Place the column into a new 2 ml collection tube (provided). Add  500ul of HB Buffer into the column. Centrifuge at 10,000 x g for 1  minute. Discard the flow-through and reuse the collection tube in next  step.

10. Place the column into a same 2 ml collection tube from step 7.  Add 700ul of DNA Wash Buffer diluted with absolute ethanol into the  column. Centrifuge at 8000 x g for 1 minute. Discard the flow-through  and reuse the collection tube in next step.

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 wash buffer must be brought to room  temperature before use.

11. Place the column into a same 2 ml collection tube from step 8.  Centrifuge at maximum for 2 minutes to completely dry the HiBind^? DNA column. Discard the flow-through and the collection tube.

12. Place the column into a sterile 1.5 ml microcentrifuge tube and  add 50-100 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 for 1 min.  Retain flowthrough containing the DNA. Place column into a second 1.5 ml  tube. Elute DNA again as step 10-11. Discard column and store the  eluted DNA at -20°C.