Vacuum／SpinProtocolforTissueDNAExtraction

實驗概要

The E.Z.N.A.^?  Tissue DNA Kit provides a rapid and easy method for the isolation of  genomic DNA for consistent PCR and Southern analysis. Up to 30 mg tissue  or up to 1 cm sections of mouse tail can be readily processed in one  time. The method can also be used for preparation of genomic DNA from  mouse tail snips, blood, buffy coat, serum, and plasma. The kit allows  single or multiple, simultaneous processing of samples. There is no need  for phenol/chloroform extractions, and timeconsuming steps such as  precipitation with isopropanol or ethanol, are eliminated. DNA purified  using the E.Z.N.A.^? Tissue DNA method is ready for applications such as PCR*, Southern blotting, and restriction digestion.

Carry out disruption, homogenization, Protease digestion, and loading onto HiBind^? DNA column as indicated previous protocols. Instead of continuing with centrifugation, follow steps blow.

實驗步驟

Note: Please read through previous section of this book before using this protocol.

1. Prepare the vacuum manifold according to manufacturer’s instruction and connect the HiBind^? DNA V-Spin column to the manifold.

2. Load the sample into HiBind^? DNA V-spin column.

3. Switch on vacuum source to draw the sample through the column and turn off the vacuum.

4. Wash the column by adding 500 ul Buffer HB, draw the wash buffer through the column by turning on the vacuum source.

5. Wash the column by adding 700 ul DNA wash buffer, draw the wash buffer through the column by turning on the vacuum source.

6. Wash the column again by adding 700 u DNA wash buffer, draw the  wash buffer through the column by turning on the vacuum source.

7. Assemble the column into a 2 ml collection tube and transfer the  column to a micro centrifuge. Spin at maxi speed (no more than 20,000 x  g) for 2 minute to dry the column.

8. Place the column in a clean 1.5 ml microcentrifuge tube and add  50-100ul DNA elution buffer. Stand for 1-2 minute and centrifuge 1  minute to elute DNA.